Monoclonal antibodies directed against trimeric forms of the hiv-1 envelope glycoprotein with broad and potent neutralizing activity

ABSTRACT

The invention provides a method for obtaining a broadly neutralizing antibody (bNab), including screening memory B cell cultures from a donor PBMC sample for neutralization activity against a plurality of HIV-1 species, cloning a memory B cell that exhibits broad neutralization activity; and rescuing a monoclonal antibody from that memory B cell culture. The resultant monoclonal antibodies are characterized by their ability to selectively bind epitopes from the Env proteins in native or monomeric form, as well as to inhibit infection of HIV-1 species from a plurality of clades. Compositions containing human monoclonal anti-HIV antibodies used for prophylaxis, diagnosis and treatment of HIV infection are provided. Methods for generating such antibodies by immunization using epitopes from conserved regions within the variable loops of gp120 are provided. Immunogens for generating anti-HIV1 bNAbs are also provided. Furthermore, methods for vaccination using suitable epitopes are provided.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/726,245 filed Mar. 17, 2010, which claims the benefit of provisionalapplications U.S. Ser. No. 61/161,010, filed Mar. 17, 2009, U.S. Ser.No. 61/165,829, filed Apr. 1, 2009, U.S. Ser. No. 61/224,739, filed Jul.10, 2009, and U.S. Ser. No. 61/285,664, filed Dec. 11, 2009, thecontents of which are each herein incorporated by reference in theirentirety.

GOVERNMENT SUPPORT

This invention was made with Government support under Grant No. AI33292awarded by the National Institutes of Health. The Government has certainrights in the invention.

INCORPORATION BY REFERENCE

The contents of the text file named “37418507001 USSeqList.txt.” whichwas created on Sep. 22, 2010 and is 125 KB in size, are herebyincorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates generally to therapy, diagnosis andmonitoring of human immunodeficiency virus (HIV) infection. Theinvention is more specifically related to human neutralizing monoclonalantibodies specific for HIV-1, such as broad and potent neutralizingmonoclonal antibodies specific for HIV-1 and their manufacture and use.Broad neutralization suggests that the antibodies can neutralize HIV-1isolates from different individuals. Such antibodies are useful inpharmaceutical compositions for the prevention and treatment of HIV, andfor the diagnosis and monitoring of HIV infection and for design of HIVvaccine immunogens.

BACKGROUND OF THE INVENTION

AIDS was first reported in the United States in 1981 and has sincebecome a major worldwide epidemic. AIDS is caused by the humanimmunodeficiency virus, or HIV. By killing or damaging cells of thebody's immune system, HIV progressively destroys the body's ability tofight infections and certain cancers. People diagnosed with AIDS may getlife-threatening diseases called opportunistic infections. Theseinfections are caused by microbes such as viruses or bacteria thatusually do not make healthy people sick. HIV is spread most oftenthrough unprotected sex with an infected partner. HIV also is spreadthrough contact with infected blood. The human immunodeficiency virus(HIV) is the cause of acquired immune deficiency syndrome (AIDS)(Barre-Sinoussi, F., et al., 1983, Science 220:868-870; Gallo, R., etal., 1984, Science 224:500-503). There are currently 1.25 million peoplein the US infected with HIV-induced acquired immunodeficiency syndromeaccording to a Center for Disease Control report. The epidemic isgrowing most rapidly among minority populations and is a leading killerof African-American males ages 25 to 44. According, AIDS affects nearlyseven times more African Americans and three times more Hispanics thanwhites. In recent years, an increasing number of African-American womenand children are being affected by HIV/AIDS. With over 40 million peopleinfected worldwide, the current global HIV pandemic ranks among thegreatest infectious disease scourges in human history.

There is therefore a need for the efficient identification andproduction of neutralizing antibodies effective against multiple cladesand strains of HIV as well as the elucidation of the target andantigenic determinants to which such antibodies bind.

SUMMARY OF THE INVENTION

The present invention provides a novel method for isolating potent,broadly neutralizing monoclonal antibodies against HIV. Peripheral BloodMononuclear Cells (PBMCs) are obtained from an HIV-infected donorselected for HIV-1 neutralizing activity in the plasma, and memory Bcells are isolated for culture in vitro. The B cell culture supernatantsare then screened by a primary neutralization assay in a high throughputformat, and B cell cultures exhibiting neutralizing activity areselected for rescue of monoclonal antibodies. It is surprisinglyobserved that neutralizing antibodies obtained by this method do notalways exhibit gp120 or gp41 binding at levels that correlate withneutralization activity. The method of the invention therefore allowsidentification of novel antibodies with cross-clade neutralizationproperties.

The present invention provides human monoclonal antibodies specificallydirected against HIV. In certain embodiments, the invention provideshuman anti-HIV monoclonal antibodies and sister clones thereof. Forinstance, an exemplary sister clone of the 1443 C16 (PG16) antibody isthe 1503 H05 (PG16) antibody, the 1456 A12 (PG16) antibody, the 1469 M23(PG16) antibody, the 1489 I13 (PG16) antibody, or the 1480_I08 (PG16)antibody.

Specifically, the invention provides an isolated anti-HIV antibody,wherein said antibody has a heavy chain with three CDRs including anamino acid sequence selected from the group consisting of the amino acidsequences of SGFTFHKYGMH (SEQ ID NO: 88), LISDDGMRKYHSDSMW (SEQ ID NO:89), and EAGGPIWHDDVKYYDFNDGYYNYHYMDV (SEQ ID NO: 6), and a light chainwith three CDRs that include an amino acid sequence selected from thegroup consisting of the amino acid sequences of NGTSSDVGGFDSVS (SEQ IDNO: 97), DVSHRPSG (SEQ ID NO: 95), and SSLTDRSHRI (SEQ ID NO: 41).

The invention provides an isolated anti-HIV antibody, wherein saidantibody has a heavy chain with three CDRs including an amino acidsequence selected from the group consisting of the amino acid sequencesof SGFTFHKYGMH (SEQ ID NO: 88), LISDDGMRKYHSDSMW (SEQ ID NO: 89), andEAGGPIWHDDVKYYDFNDGYYNYHYMDV (SEQ ID NO: 6), and a light chain withthree CDRs that include an amino acid sequence selected from the groupconsisting of the amino acid sequences of NGTRSDVGGFDSVS (SEQ ID NO:92), DVSHRPSG (SEQ ID NO: 95), and SSLTDRSHRI (SEQ ID NO: 41).

The invention provides an isolated anti-HIV antibody, wherein saidantibody has a heavy chain with three CDRs including an amino acidsequence selected from the group consisting of the amino acid sequencesof SGFTFHKYGMH (SEQ ID NO: 88), LISDDGMRKYHSDSMW (SEQ ID NO: 89), andEAGGPIWHDDVKYYDFNDGYYNYHYMDV (SEQ ID NO: 6), and a light chain withthree CDRs that include an amino acid sequence selected from the groupconsisting of the amino acid sequences of NGTSRDVGGFDSVS (SEQ ID NO:93), DVSHRPSG (SEQ ID NO: 95), and SSLTDRSHRI (SEQ ID NO: 41).

The invention provides an isolated anti-HIV antibody, wherein saidantibody has a heavy chain with three CDRs including an amino acidsequence selected from the group consisting of the amino acid sequencesof SGFTFHKYGMH (SEQ ID NO: 88), LISDDGMRKYHSNSMW (SEQ ID NO: 98), andEAGGPIWHDDVKYYDFNDGYYNYHYMDV (SEQ ID NO: 6), and a light chain withthree CDRs that include an amino acid sequence selected from the groupconsisting of the amino acid sequences of NGTSSDVGGFDSVS (SEQ ID NO:97), DVSHRPSG (SEQ ID NO: 95), and SSLTDRSHRI (SEQ ID NO: 41).

The invention provides an isolated anti-HIV antibody, wherein saidantibody has a heavy chain with three CDRs including an amino acidsequence selected from the group consisting of the amino acid sequencesof SGGTFSSYAFT (SEQ ID NO: 104), MVTPIFGEAKYSQRFE (SEQ ID NO: 105), andRAVPIATDNWLDP (SEQ ID NO: 102), and a light chain with three CDRs thatinclude an amino acid sequence selected from the group consisting of theamino acid sequences of RASQTINNYLN (SEQ ID NO: 107), GASNLQNG (SEQ IDNO: 108), and QQSFSTPRT (SEQ ID NO: 42).

The invention provides an isolated anti-HIV antibody, wherein saidantibody has a heavy chain with three CDRs including an amino acidsequence selected from the group consisting of the amino acid sequencesof SGGTFSSYAFT (SEQ ID NO: 104), MVTPIFGEAKYSQRFE (SEQ ID NO: 105), andRRAVPIATDNWLDP (SEQ ID NO: 103), and a light chain with three CDRs thatinclude an amino acid sequence selected from the group consisting of theamino acid sequences of RASQTINNYLN (SEQ ID NO: 107), GASNLQNG (SEQ IDNO: 108), and QQSFSTPRT (SEQ ID NO: 42).

The invention provides an isolated anti-HIV antibody, wherein saidantibody has a heavy chain with three CDRs including an amino acidsequence selected from the group consisting of the amino acid sequencesof SGGAFSSYAFS (SEQ ID NO: 110), MITPVFGETKYAPRFQ (SEQ ID NO: 111), andRAVPIATDNWLDP (SEQ ID NO: 102), and a light chain with three CDRs thatinclude an amino acid sequence selected from the group consisting of theamino acid sequences of RASQTIHTYL (SEQ ID NO: 113), GASTLQSG (SEQ IDNO: 114), and QQSYSTPRT (SEQ ID NO: 43).

The invention provides an isolated anti-HIV antibody, wherein saidantibody has a heavy chain with three CDRs including an amino acidsequence selected from the group consisting of the amino acid sequencesof SGGAFSSYAFS (SEQ ID NO: 110), MITPVFGETKYAPRFQ (SEQ ID NO: 111), andRRAVPIATDNWLDP (SEQ ID NO: 103), and a light chain with three CDRs thatinclude an amino acid sequence selected from the group consisting of theamino acid sequences of RASQTIHTYL (SEQ ID NO: 113), GASTLQSG (SEQ IDNO: 114), and QQSYSTPRT (SEQ ID NO: 43).

The invention provides an isolated anti-HIV antibody, wherein saidantibody has a heavy chain with three CDRs including an amino acidsequence selected from the group consisting of the amino acid sequencesof SGYSFIDYYLH (SEQ ID NO: 116), LIDPENGEARYAEKFQ (SEQ ID NO: 117).AVGADSGSWFDP (SEQ ID NO: 118), and a light chain with three CDRs thatinclude an amino acid sequence selected from the group consisting of theamino acid sequences of SGSKLGDKYVS (SEQ ID NO: 120), ENDRRPSG (SEQ IDNO: 121), QAWETITTFVF (SEQ ID NO: 44).

The invention provides an isolated anti-HIV antibody, wherein saidantibody has a heavy chain with three CDRs including an amino acidsequence selected from the group consisting of the amino acid sequencesof SGFDFSRQGMH (SEQ ID NO: 123), FIKYDGSEKYHADSVW (SEQ ID NO: 124), andEAGGPDYRNGYNYYDFYDGYYNYHYMDV (SEQ ID NO: 7), and a light chain withthree CDRs that include an amino acid sequence selected from the groupconsisting of the amino acid sequences of NGTSNDVGGYESVS (SEQ ID NO:126), DVSKRPSG (SEQ ID NO: 127), and KSLTSTRRRV (SEQ ID NO: 45).

The invention provides an isolated anti-HIV antibody, wherein saidantibody has a heavy chain with three CDRs including an amino acidsequence selected from the group consisting of the amino acid sequencesof SGFTFHKYGMH (SEQ ID NO: 88), LISDDGMRKYHSDSMW (SEQ ID NO: 89),EAGGPIWHDDVKYYDFNDGYYNYHYMDV (SEQ ID NO: 6), SGGTFSSYAFT (SEQ ID NO:104), MVTPIFGEAKYSQRFE (SEQ ID NO: 105), RAVPIATDNWLDP (SEQ ID NO: 102),SGGAFSSYAFS (SEQ ID NO: 110), MITPVFGETKYAPRFQ (SEQ ID NO: 111),SGYSFIDYYLH (SEQ ID NO: 116), LIDPENGEARYAEKFQ (SEQ ID NO: 117),AVGADSGSWFDP (SEQ ID NO: 118), SGFDFSRQGMH (SEQ ID NO: 123),FIKYDGSEKYHADSVW (SEQ ID NO: 124), EAGGPDYRNGYNYYDFYDGYYNYHYMDV (SEQ IDNO: 7), LISDDGMRKYHSNSMW (SEQ ID NO: 98), wherein said antibody binds toand neutralizes HIV-1.

The invention provides an isolated anti-HIV antibody, wherein saidantibody has a light chain with three CDRs that include an amino acidsequence selected from the group consisting of the amino acid sequencesof NGTSSDVGGFDSVS (SEQ ID NO: 97), DVSHRPSG (SEQ ID NO: 95), SSLTDRSHRI(SEQ ID NO: 41), RASQTINNYLN (SEQ ID NO: 107), GASNLQNG (SEQ ID NO:108), QQSFSTPRT (SEQ ID NO: 42), RASQTIHTYL (SEQ ID NO: 113), GASTLQSG(SEQ ID NO: 114), QQSYSTPRT (SEQ ID NO: 43), SGSKLGDKYVS (SEQ ID NO:120), ENDRRPSG (SEQ ID NO: 121), QAWETITTTFVF (SEQ ID NO: 44),NGTSNDVGGYESVS (SEQ ID NO: 126), DVSKRPSG (SEQ ID NO: 127), KSLTSTRRRV(SEQ ID NO: 45), NGTRSDVGGFDSVS (SEQ ID NO: 92), NGTSRDVGGFDSVS (SEQ IDNO: 93), wherein said antibody binds to and neutralizes HIV-1.

The invention provides an isolated anti-HIV antibody, wherein saidantibody has a heavy chain with three CDRs including an amino acidsequence selected from the group consisting of the amino acid sequencesof SGFTFHKYGMH (SEQ ID NO: 88), LISDDGMRKYHSDSMW (SEQ ID NO: 89),EAGGPIWHDDVKYYDFNDGYYNYHYMDV (SEQ ID NO: 6), SGGTFSSYAFT (SEQ ID NO:104), MVTPIFGEAKYSQRFE (SEQ ID NO: 105), RRAVPIATDNWLDP (SEQ ID NO:103), SGGAFSSYAFS (SEQ ID NO: 110), MITPVFGETKYAPRFQ (SEQ ID NO: 111),SGYSFIDYYLH (SEQ ID NO: 116), LIDPENGEARYAEKFQ (SEQ ID NO: 117),AVGADSGSWFDP (SEQ ID NO: 118), SGFDFSRQGMH (SEQ ID NO: 123),FIKYDGSEKYHADSVW (SEQ ID NO: 124), EAGGPDYRNGYNYYDFYDGYYNYHYMDV (SEQ IDNO: 7). LISDDGMRKYHSNSMW (SEQ ID NO: 98), wherein said antibody binds toand neutralizes HIV-1.

The invention provides an isolated anti-HIV antibody, wherein saidantibody has a light chain with three CDRs that include an amino acidsequence selected from the group consisting of the amino acid sequencesof NGTSSDVGGFDSVS (SEQ ID NO: 97), DVSHRPSG (SEQ ID NO: 95), SSLTDRSHRI(SEQ ID NO: 41), RASQTINNYLN (SEQ ID NO: 107), GASNLQNG (SEQ ID NO:108), QQSFSTPRT (SEQ ID NO: 42), RASQTIHTYL (SEQ ID NO: 113), GASTLQSG(SEQ ID NO: 114), QQSYSTPRT (SEQ ID NO: 43), SGSKLGDKYVS (SEQ ID NO:120), ENDRRPSG (SEQ ID NO: 121), QAWETTTTTFVF (SEQ ID NO: 44),NGTSNDVGGYESVS (SEQ ID NO: 126), DVSKRPSG (SEQ ID NO: 127), KSLTSTRRRV(SEQ ID NO: 45), NGTRSDVGGFDSVS (SEQ ID NO: 92), NGTSRDVGGFDSVS (SEQ IDNO: 93), wherein said antibody binds to and neutralizes HIV-1.

The invention provides an isolated anti-HIV antibody or fragmentthereof, wherein said antibody includes: (a) a V_(H) CDR1 regioncomprising the amino acid sequence of SEQ ID NO: 88, 104, 110, 116, or123; (b) a V_(H) CDR2 region comprising the amino acid sequence of SEQID NO: 98, 89, 91, 105, 111, 117, or 124; and (c) a V_(H) CDR3 regioncomprising the amino acid sequence of SEQ ID NO: 6, 102, 103, 118, or 7,wherein said antibody binds to and neutralizes HIV-1. In certainaspects, this antibody further includes: (a) a V_(L) CDR1 regioncomprising the amino acid sequence of SEQ ID NO: 93, 92, 97, 94, 107,113, 120, or 126; (b) a V_(L) CDR2 region comprising the amino acidsequence of SEQ ID NO: 95, 108, 114, 121, or 127; and (c) a V_(L) CDR3region comprising the amino acid sequence of SEQ ID NO: 41, 42, 43, 44,or 45.

The invention provides an isolated fully human monoclonal anti-HIVantibody including: a) a heavy chain sequence comprising the amino acidsequence of SEQ ID NO: 31 and a light chain sequence comprising aminoacid sequence SEQ ID NO: 32, or b) a heavy chain sequence comprising theamino acid sequence of SEQ ID NO: 33 and a light chain sequencecomprising amino acid sequence SEQ ID NO: 34, or c) a heavy chainsequence comprising the amino acid sequence of SEQ ID NO: 35 and a lightchain sequence comprising amino acid sequence SEQ ID NO: 36, or d) aheavy chain sequence comprising the amino acid sequence of SEQ ID NO: 37and a light chain sequence comprising amino acid sequence SEQ ID NO: 38,or e) a heavy chain sequence comprising the amino acid sequence of SEQID NO: 39 and a light chain sequence comprising amino acid sequence SEQID NO: 40, or f) a heavy chain sequence comprising the amino acidsequence of SEQ ID NO: 140 and a light chain sequence comprising aminoacid sequence SEQ ID NO: 96, or g) a heavy chain sequence comprising theamino acid sequence of SEQ ID NO: 48 and a light chain sequencecomprising amino acid sequence SEQ ID NO: 51, or h) a heavy chainsequence comprising the amino acid sequence of SEQ ID NO: 54 and a lightchain sequence comprising amino acid sequence SEQ ID NO: 57, or i) aheavy chain sequence comprising the amino acid sequence of SEQ ID NO: 60and a light chain sequence comprising amino acid sequence SEQ ID NO: 32.

The invention provides a composition including any one of the isolatedanti-HIV antibodies described herein.

Optionally, an anti-HIV human monoclonal antibody of the invention isisolated from a B-cell from an HIV-1-infected human donor. In someembodiments, the antibody is effective in neutralizing a plurality ofdifferent clades of HIV. In some embodiments, the antibody is effectivein neutralizing a plurality of different strain within the same clade ofHIV-1. In some embodiments, the neutralizing antibody binds to the HIVenvelope proteins gp120, or gp41 or envelope protein on HIV-1pseudovirions or expressed on transfected or infected cell surfaces. Insome embodiments, the neutralizing antibody does not bind to recombinantor monomeric envelope proteins gp120, or gp41 or envelope protein onHIV-1 pseudovirions or expressed on transfected or infected cellsurfaces but binds to natural trimeric forms of the HIV-1 Env proteins.

The present invention provides human monoclonal antibodies wherein theantibodies are potent, broadly neutralizing antibody (bNAb). In someembodiments, a broadly neutralizing antibody is defined as a bNAb thatneutralizes HIV-1 species belonging to two or more different clades. Insome embodiments the different clades are selected from the groupconsisting of clades A, B, C, D, E, AE, AG, G or F. In some embodimentsthe HIV-1 strains from two or more clades comprise virus from non-Bclades.

In some embodiments, a broadly neutralizing antibody is defined as abNAb that neutralizes at least 60% of the HIV-1 strains listed in Tables18A-18F. In some embodiments, at least 70%, or at least 80%, or at least90% of the HIV-1 strains listed in Tables 18A-18F are neutralized.

In some embodiments, a potent, broadly neutralizing antibody is definedas a bNAb that displays a potency of neutralization of at least aplurality of HIV-1 species with an IC50 value of less than 0.2 μg/mL. Insome embodiments the potency of neutralization of the HIV-1 species hasan IC50 value of less than 0.15 μg/mL, or less than 0.10 μg/mL, or lessthan 0.05 μg/mL. A potent, broadly neutralizing antibody is also definedas a bNAb that displays a potency of neutralization of at least aplurality of HIV-1 species with an IC90 value of less than 2.0 μg/mL. Insome embodiments the potency of neutralization of the HIV-1 species hasan IC90 value of less than 1.0 μg/mL, or less than 0.5 μg/mL.

Exemplary monoclonal antibodies that neutralize HIV-1 include 1496_C09(PG9), 1443_C16 (PG16), 1456_P20 (PG20), 1460_G14 (PGG14), and 1495_C14(PGC14) described herein. Alternatively, the monoclonal antibody is anantibody that binds to the same epitope as 1496_C09 (PG9), 1443_C16(PG16), 1456_P20 (PG20), 1460_G14 (PGG14), and 1495_C14 (PGC14).Specifically, monoclonal antibodies PG9 and PG16 are broad and potentneutralizing antibodies. The antibodies are respectively referred toherein as HIV antibodies.

The invention provides a number of isolated human monoclonal antibodies,wherein each said monoclonal antibody binds to HIV-1 infected ortransfected cells; and binds to HIV-1 virus. A neutralizing antibodyhaving potency in neutralizing HIV-1, or a fragment thereof is provided.In some embodiments a neutralizing antibody of the invention exhibitshigher neutralization index and/or a higher affinity for binding to theenvelope proteins gp120, or gp41 than anti-HIV mAbs known in the art,such as the mAb b12. (Burton D R et al., Science Vol. 266. no. 5187, pp.1024-1027). Exemplary monoclonal antibodies 1496_C09 (PG9), 1443_C16(PG16), 1456_P20 (PG20), 1460_G14 (PGG14), and 1495_C14 (PGC14) exhibitbinding to the envelope glycoprotein gp120, but not gp41, in an ELISAassay, however gp20 binding does not always correlate withneutralization activity against specific strains of HIV-1. In someembodiments, monoclonal antibodies, for example 1443_C16 (PG16) and1496_C09 (PG9), display none or weak gp120 binding activity against aparticular strain but bind to HIV-1 trimer on transfected or infectedcell surface and/or virion and exhibit broad and potent neutralizationactivity against that strain of HIV-1.

In one aspect the antibody is a monoclonal antibody comprising one ormore polypeptides selected from the group consisting of 1496_C09 (PG9),1443_C16 (PG16), 1456_P20 (PG20), 1460_G14 (PGG14), and 1495_C14(PGC14); comprising a heavy chain selected from the group consisting ofthe heavy chain of 1496_C09 (PG9), 1443_C16 (PG16), 1456_P20 (PG20),1460_G14 (PGG14), and 1495_C14 (PGC14); comprising a heavy chaincomprising a CDR selected from the group consisting of the CDRs of theheavy chain of 1496_C09 (PG9), 1443_C16 (PG16), 1456_P20 (PG20),1460_G14 (PGG14), and 1495_C14 (PGC14); comprising a light chainselected from the group consisting of the light chain of 1496_C09 (PG9),1443_C16 (PG16), 1456_P20 (PG20), 1460_G14 (PGG14), and 1495_C14(PGC14); comprising a light chain comprising a CDR selected from thegroup consisting of the CDRs of the light chain of 1496_C09 (PG9),1443_C16 (PG16), 1456_P20 (PG20), 1460_G14 (PGG14), and 1495_C14(PGC14).

The invention relates to an antibody or a fragment thereof, such as Fab,Fab′, F(ab′)2 and Fv fragments that binds to an epitope or immunogenicpolypeptide capable of binding to an antibody selected from 1496_C09(PG9), 1443_C16 (PG16), 1456_P20 (PG20), 1460_G14 (PGG14), and 1495_C14(PGC14). The invention also relates to immunogenic polypeptides encodingsuch epitopes.

Nucleic acid molecules encoding such antibodies, and vectors and cellscarrying such nucleic acids are also provided.

The invention relates to a pharmaceutical composition comprising atleast one antibody or fragment as recited herein, together with apharmaceutically acceptable carrier.

The invention relates to a method of immunizing, preventing orinhibiting HIV infection or an HIV-related disease comprising the stepsof identifying a patient in need of such treatment and administering tosaid patient a therapeutically effective amount of at least onemonoclonal antibody as recited herein.

In a further aspect the HIV antibodies according to the invention arelinked to a therapeutic agent or a detectable label.

Additionally, the invention provides methods for stimulating an immuneresponse, treating, preventing or alleviating a symptom of an HIV viralinfection by administering an HIV antibody to a subject

In another aspect, the invention provides methods of administering theHIV antibody of the invention to a subject prior to, and/or afterexposure to an HIV virus. For example, the HIV antibody of the inventionis used to treat or prevent HIV infection. The HIV antibody isadministered at a dose sufficient to promote viral clearance oreliminate HIV infected cells.

Also included in the invention is a method for determining the presenceof an HIV virus infection in a patient, by contacting a biologicalsample obtained from the patient with an HIV antibody; detecting anamount of the antibody that binds to the biological sample; andcomparing the amount of antibody that binds to the biological sample toa control value.

The invention further provides a diagnostic kit comprising an HIVmonoclonal antibody.

The invention relates to a broadly neutralizing antibody (bNAb) whereinthe antibody neutralizes at least one member of each clade with apotency greater than that of the bNAbs b12, 2G12, 2F5 and 4E10respectively.

The invention relates to a broadly neutralizing antibody (bNAb) whereinthe antibody does not bind monomeric gp120 or gp41 proteins of the HIV-1env gene. The antibody binds with higher affinity to trimeric forms ofthe HIV-1 Env expressed on a cell surface than to the monomeric gp120 orartificially trimerized gp140. In some aspects, the antibody binds withhigh affinity to uncleaved HIV-1 gp160 trimers on a cell surface.

The invention relates to a broadly neutralizing antibody (bNAb) whereinthe antibody binds an epitope within the variable loop of gp120, whereinthe epitope comprises the conserved regions of V2 and V3 loops of gp120,wherein the epitope comprises N-glycosylation site at residue Asn-160within the V2 loop of gp120, wherein the antibody binds an epitopepresented by a trimeric spike of gp120 on a cell surface, wherein theepitope is not presented when gp120 is artificially trimerized. In someembodiments, the antibody does not neutralize the HIV-1 in the absenceof N-glycosylation site at residue Asn-160 within the V2 loop of gp120.

The invention relates to a broadly neutralizing antibody (bNAb) selectedfrom the group consisting of PG16 and PG9.

The invention relates to an antigen or an immunogenic polypeptide, or avaccine comprising such antigen or immunogenic polypeptide, forproducing a broadly neutralizing antibody (bNAb) by an immune response,the antigen comprising an epitope within the variable loop of gp120according to the invention.

The invention relates to method for passive or active immunization of anindividual against a plurality of HIV-1 species across one or moreclades, the method comprising: providing a broadly neutralizing antibody(bNAb) wherein the bNAb neutralizes HIV-1 species belonging to two ormore clades, and further wherein the potency of neutralization of atleast one member of each clade is determined by an IC50 value of lessthan 0.005 μg/mL. In some embodiments, the antibody is selected from thegroup consisting of PG9 and PG16.

In some embodiments, the antibody is produced by active immunizationwith an antigen comprising an epitope within the variable loop of gp120,wherein the epitope comprises the conserved regions of V2 and V3 loopsof gp120 or, wherein the epitope comprises an N-glycosylation site atresidue Asn-160 within the V2 loop of gp120. In some aspects, theepitope is presented by a trimeric spike of gp120 on a cell surface, andthe epitope is not presented when gp120 is monomeric or artificiallytrimerized.

Other features and advantages of the invention will be apparent from andare encompassed by the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic tree diagram of Clustal W-aligned variable regionsequences of heavy chains of the monoclonal antibodies.

FIG. 1B is a schematic tree diagram of Clustal W-aligned variable regionsequences of light chains of the monoclonal antibodies.

FIG. 2 is a flow chart of the process for isolation of monoclonalantibodies according to the invention.

FIG. 3A is a schematic diagram that summarizes the screening results forneutralization and HIV-env protein (gp120 and gp41) binding assays fromwhich B cell cultures were selected for antibody rescue and themonoclonal antibodies 1496_C09 (PG9), 1443_C16 (PG16), 1456_P20 (PG20),1460_G14 (PGG14), and 1495_C14 (PGC14) were derived. A neutralizationindex value of 1.5 was used as a cut-off.

FIG. 3B is a schematic diagram that summaries the neutralizing activityand HIV-env protein (gp120 and gp41) binding activities of themonoclonal antibodies 1496_C09 (PG9), 1443_C16 (PG16), 1456_P20 (PG20),1460_G14 (PGG14), and 1495_C14 (PGC14) as determined by ELISA assaysamong the B cell supernatants using a neutralization index cut-off valueof 2.0. The neutralization index was expressed as the ratio ofnormalized relative luminescence units (RLU) of SIVmac239 to that oftest viral strain derived from the same test B cell culture supernatant.The cut-off values used to distinguish neutralizing hits were determinedby the neutralization index of a large number of negative control wellscontaining B cell culture supernatants derived from healthy donors.

FIG. 4A-B is a series of graphs depicting the neutralization activity ofmonoclonal antibodies 1443_C16 (PG16) and 1496_C09 (PG9) to additionalpseudoviruses not included in Tables 17A and 17B.

FIG. 5 is a graph depicting the dose response curves of 1456_P20 (PG20),1495_C14 (PGC14) and 1460_G14 (PGG14) binding to recombinant gp120 inELISA as compared to control anti-gp120 (b12). Data is presented asaverage OD values of triplicate ELISA wells obtained on the same plate.

FIG. 6A-C is a series of graphs depicting the results from ELISA bindingassays of monoclonal antibodies 1443_C16 (PG16) and 1496_C09 (PG9) toHIV-1 YU2 gp140, JR-CSFgp120, membrane-proximal external regions (MPER)peptide of gp41 and V3 polypeptide.

FIG. 7 is a graph depicting the results of a binding assay usingmonoclonal antibodies 1443_C16 (PG16) and 1496_C09 (PG9) to HIV-1 YU2gp160 expressed on the cell surface in the presence and absence ofsoluble CD4 (sCD4).

FIG. 8A-B is a graph depicting the results of a binding assay usingmonoclonal antibodies 1443_C16 (PG16) and 1496_C09 (PG9) to HIV-1 gp160transfected cells.

FIG. 9 is a series of graphs depicting the results of a capture assay.The data describe capturing of entry-competent JRCSF pseudovirus byneutralizing monoclonal antibodies 1443_C16 (PG16) and 1496_C09 (PG9) ina dose-dependent manner.

FIG. 10A is a graph depicting the results of a competitive binding assayusing monoclonal antibodies sCD4, PG16 and PG9, wherein the claimedantibodies compete for the binding of monoclonal antibody 1443_C16(PG16) to pseudovirus but control antibodies b12, 2G12, 2F5 and 4E10 donot competitively bind to the pseudovirus.

FIG. 10B is a graph depicting the results of a competitive binding assayusing monoclonal antibodies sCD4, PG16 and PG9, wherein the claimedantibodies compete for the binding of monoclonal antibody 1496_C09 (PG9)to pseudovirus but control antibodies b12, 2G12, 2F5 and 4E10 do notcompetitively bind to the pseudovirus.

FIG. 11A is a series of graphs depicting the results of a binding assayusing PG9 and PG16. The data show that PG9 and PG16 bind to monomericgp120 and artificially trimerized gp140 constructs as determined byELISA. IgG b12 was used as a control for ELISA assays.

FIG. 11B is a series of graphs depicting the results of a binding assayusing PG9 and PG16. The data show that PG9 and PG116 bind to Envexpressed on the surface of 293T cells as determined by flow cytometry.The bNAb b12 and the non-neutralizing antibody b6 are included in thecell surface binding assays to show the expected percentages of cleavedand uncleaved Env expressed on the cell surface.

FIG. 12 is a series of graphs depicting the results of a binding assayusing PG9 and PG16 and cleavage-defective HIV-1YU2 trimers. PG9 and PG16bind with high affinity to cleavage-defective HIV-1YU2 trimers asdetermined by flow cytometry. Binding curves were generated by plottingthe MFI of antigen binding as a function of antibody concentration.

FIG. 13A-E is a series of graphs depicting the mapping the PG9 and PG16epitopes. Competitor antibody is indicated at the top of each graph.2G12 is included to control for cell surface Env expression. A: PG9 andPG16 compete with each other for cell surface Env binding and neitherantibody competes with the CD4bs antibody b12 for Env binding. B:Ligation of cell surface Env with sCD4 diminishes binding of PG9 andPG16. 2G12 is included to control for CD4-induced shedding of gp120. C:sCD4 inhibits binding of PG9 to artificially trimerized gp140YU-2 asdetermined by ELISA. D: PG9 competes with 10/76b (anti-V2), F425/b4e8(anti-V3) and X5 (CD4i) for gp120 binding in competition ELISA assays.E: PG9 and PG16 fail to bind variable loop deleted HIV-1JR-CSF variantsexpressed on the surface of 293T cells.

FIG. 14 is a series of graphs depicting the results of competition ELISAassays using the monoclonal antibody PG9.

FIG. 15 is a graph depicting monoclonal antibody binding, PG9 or PG16,to HIV-1JR-FLΔCT E168K Env expressed on the surface of 293T cells asdetermined by flow cytometry.

FIG. 16 is a graph depicting monoclonal antibody PG9 binding todeglycosylated gp20.

FIG. 17 is a series of graphs depicting the neutralization activity ofPG9 and PG16 against HIV-1SF162 and HIV-1SF162 K160N, which wasdetermined using a single-round replication luciferase reporter assay ofpseudotyped virus.

FIG. 18 is a series of graphs depicting the binding of PG9 and PG16 tomixed trimers. Alanine substitutions at positions 160 and 299 wereintroduced into HIV-1YU2 Env to abolish binding of PG9 and PG16. Analanine substitution at position 295 was also introduced into the sameconstruct to abrogate binding of 2G12. Co-transfection of 293T cellswith WT and mutant plasmids in a 1:2 ratio resulted in the expression of29% mutant homotrimers, 44% heterotrimers with two mutant subunits, 23%heterotrimers with one mutant subunit, and 4% wild-type homotrimers.

FIG. 19 is a series of graphical depictions of the number of nucleotideor amino acid differences in the heavy chain sequences of sister clonesof 1443 C16 (PG16) among each other. Note that the single nucleotidedifference of 1408 I08 translates into an identical protein sequence of1443 C16. The nucleotide sequence of the 1408 I08 light chain isidentical to the nucleotide sequence of the light chain of 1443 C16.

FIG. 20A is a tree diagram illustrating the correlation of the heavychain of 1443 C16 sister clones to the heavy chain of 1496 C09 at thenucleotide level.

FIG. 20B is a tree diagram illustrating the correlation of the lightchain of 1443 C16 sister clones to the light chain of 1496 C09 at thenucleotide level.

FIG. 21A is a tree diagram illustrating the correlation of the heavychain of 1443 C16 sister clones to the heavy chain of 1496 C09 at theprotein level.

FIG. 21B is a tree diagram illustrating the correlation of the lightchain of 1443 C16 sister clones to the light chain of 1496 C09 at theprotein level.

DETAILED DESCRIPTION OF THE INVENTION

In the sera of human immunodeficiency virus type 1 (HIV-1) infectedpatients, anti-virus antibodies can be detected over a certain periodafter infection without any clinical manifestations of the acquiredimmunodeficiency syndrome (AIDS). At this state of active immuneresponse, high numbers of antigen-specific B-cells are expected in thecirculation. These B-cells are used as fusion partners for thegeneration of human monoclonal anti-HIV antibodies. One major drawbackto finding a vaccine composition suitable for more reliable preventionof human individuals from HIV-1 infection and/or for more successfultherapeutic treatment of infected patients is the ability of the HIV-1virus to escape antibody capture by genetic variation, which very oftenrenders the remarkable efforts of the researchers almost useless. Suchescape mutants may be characterized by a change of only one or severalof the amino acids within one of the targeted antigenic determinants andmay occur, for example, as a result of spontaneous or induced mutation.In addition to genetic variation, certain other properties of the HIV-1envelope glycoprotein makes it difficult to elicit neutralizingantibodies making generation of undesirable non-neutralizing antibodiesa major concern (see Phogat S K and Wyatt R T, Curr Pharm Design 2007;13(2):213-227).

HIV-1 is among the most genetically diverse viral pathogens. Of thethree main branches of the HIV-1 phylogenetic tree, the M (main), N(new), and O (outlier) groups, group M viruses are the most widespread,accounting for over 99% of global infections. This group is presentlydivided into nine distinct genetic subtypes, or clades (A through K),based on full-length sequences. Env is the most variable HIV-1 gene,with up to 35% sequence diversity between clades, 20% sequence diversitywithin clades, and up to 10% sequence diversity in a single infectedperson (Shankarappa, R. et al. 1999. J. Virol. 73:10489-10502). Clade Bis dominant in Europe, the Americas, and Australia. Clade C is common insouthern Africa, China, and India and presently infects more peopleworldwide than any other clade (McCutchan, F E. 2000. Understanding thegenetic diversity of HIV-1. AIDS 14(Suppl. 3):S31-S44). Clades A and Dare prominent in central and eastern Africa.

Neutralizing antibodies (NAbs) against viral envelope proteins (Env)provide adaptive immune defense against human immunodeficiency virustype 1 (HIV-1) exposure by blocking the infection of susceptible cells(Kwong P D et al., 2002. Nature 420: 678-682). The efficacy of vaccinesagainst several viruses has been attributed to their ability to elicitNAbs. However, despite enormous efforts, there has been limited progresstoward an effective immunogen for HIV-1. (Burton, D. R. 2002. Nat. Rev.Immunol. 2:706-713).

HIV-1 has evolved with an extensive array of strategies to evadeantibody-mediated neutralization. (Barouch, D. H. Nature 455, 613-619(2008); Kwong, P. D. & Wilson. I. A. Nat Immunol 10, 573-578 (2009);Karlsson Hedestam, G. B., et al. Nat Rev Microbiol 6, 143-155 (2008)).However, broadly neutralizing antibodies (bNAbs) develop over time in aproportion of HIV-1 infected individuals. (Leonidas Stamatatos, L. M.,Dennis R Burton, and John Mascola. Nature Medicine (E-Pub: Jun. 14,2009); PMID: 19525964.) A handful of broadly neutralizing monoclonalantibodies have been isolated from clade B infected donors. (Burton, D.R., et al. Science 266, 1024-1027 (1994); Trkola, A., et al. J Virol 69,6609-6617 (1995); Stiegler, G., et al. AIDS Res Hum Retroviruses 17,1757-1765 (2001)). These antibodies tend to display less breadth andpotency against non-clade B viruses, and they recognize epitopes on thevirus that have so far failed to elicit broadly neutralizing responseswhen incorporated into a diverse range of immunogens. (Phogat, S. &Wyatt, R. Curr Pharm Design 13, 213-227 (2007); Montero, M., van Houten,N. E., Wang, X. & Scott, J. K. Microbiol Mol Biol Rev 72, 54-84, tableof contents (2008); Scanlan, C. N., Offer, J., Zitzmann, N. & Dwek, R.A. Nature 446, 1038-1045 (2007)). Despite the enormous diversity of thehuman immunodeficiency virus (HIV), all HIV viruses known to dateinteract with the same cellular receptors (CD4 and/or a co-receptor,CCR5 or CXCR4). Most neutralizing antibodies bind to functional regionsinvolved in receptor interactions and cell membrane fusion. However, thevast majority of neutralizing antibodies isolated to date do notrecognize more than one clade, therefore exhibiting limited protectiveefficacy in vitro or in vivo. (See Binley J M et al., 2004. J. Virol.78(23):13232-13252). The rare broadly neutralizing human monoclonalantibodies (mAbs) that have been isolated from HIV+ clade B-infectedhuman donors bind to products of the env gene of HIV-1, gp120 and thetransmembrane protein gp41. (Parren, P W et al. 1999. AIDS13:S137-S162). However, a well-known characteristic of the HIV-1envelope glycoprotein is its extreme variability. It has been recognizedthat even relatively conserved epitopes on HIV-1, such as the CD4binding site, show some variability between different isolates(Poignard. P., et al., Ann. Rev. Immunol. (2001) 19:253-274). Even anantibody targeted to one of these conserved sites can be expected tosuffer from a reduced breadth of reactivity across multiple differentisolates.

The few cross-clade reactive monoclonal antibodies known to date havebeen isolated by processes involving generation of panels of specificviral antibodies from peripheral blood lymphocytes (PBLs) ofHIV-infected individuals, either via phage display, or via conventionalimmortalization techniques such as hybridoma or Epstein Barr virustransformation, electrofusion and the like. These are selected based onreactivity in vitro to HIV-1 proteins, followed by testing for HIVneutralization activity.

An antibody phage surface expression system was used to isolate thecross-clade neutralizing Fab (fragment, antigen binding) b12 occurringin a combinatorial library. The Fab b12 was screened by panning forenvelope glycoprotein gp120 binding activity and neutralizing activityagainst the HIV-1 (HXBc2) isolate was observed. (Roben P et al., J.Virol. 68(8): 4821-4828(1994); Barbas C F et al., Proc. Natl. Acad. Sci.USA Vol. 89, pp. 9339-9343, (1992); Burton D P et al., Proc. Natl. Acad.Sci. USA Vol. 88, pp. 10134-10137 (1991)).

Human B cell immortalization was used to isolate the cross-cladeneutralizing monoclonal antibodies 2G12, 2F5, and 4E10 from HIV-infectedindividuals. The monoclonal antibody 2G12 binds to a glycotope on thegp120 surface glycoprotein of HIV-1 and had been shown to display broadneutralizing patterns. (Trkola A., et al., J. Virol. 70(2): 1100-1108(1996), Buchacher, A., et al., 1994. AIDS Res. Hum. Retroviruses10:359-369). The monoclonal antibody 2F5 which had been shown to bind asequence within the external domain of the gp41 envelope glycoprotein ofHIV-1 was found to have broad neutralization properties. (Conley A JProc. Natl. Acad. Sci. USA Vol. 91, pp. 3348-3352 (1994); Muster T etal., J. Virol. 67(11):6642-6647 (1993); Buchacher A et al., 1992,Vaccines 92:191-195). The monoclonal antibody 4E10, which binds to anovel epitope C terminal of the ELDKWA sequence in gp41 recognized by2F5, has also been found to have potent cross-clade neutralizationactivity. (Buchacher, A., et al., 1994. AIDS Res. Hum. Retroviruses10:359-369; Stiegler, G., et al., 2001. AIDS Res. Hum. Retroviruses17(18):1757-1765)).

Other studies on antibody neutralization of HIV-1 (Nara, P. L., et al.(1991) FASEB J. 5:2437-2455.) focused on a single linear epitope in thethird hypervariable region of the viral envelope glycoprotein gp120known as the V3 loop. Antibodies to this loop are suggested toneutralize by inhibiting fusion of viral and cell membranes. Howeverthere is sequence variability within the loop and neutralizingantibodies are sensitive to sequence variations outside the loop (AlbertJ. et al., (1990) AIDS 4, 107-112). Hence anti-V3 loop antibodies areoften strain-specific and mutations in the loop in vivo may provide amechanism for viral escape from antibody neutralization. There is someindication that not all neutralizing antibodies act by blocking theattachment of virus, since a number of mouse monoclonal antibodiesinhibiting CD4 binding to gp120 are either non-neutralizing (Lasky L A,et al., (1987) Cell 50:975-985.) or only weakly neutralizing (Sun N., etal., (1989) J. Virol. 63, 3579-3585).

It is widely accepted that such a vaccine will require both T-cellmediated immunity as well as the elicitation of a broadly neutralizingantibody (bNAb) response. (Barouch, D. H. Nature 455, 613-619 (2008);Walker, B. D. & Burton, D. R. Science 320, 760-764 (2008); Johnston, M.I. & Fauci, A. S. N Engl J Med 356, 2073-2081 (2007)). All of the knownbNAbs provide protection in the best available primate models (Veazey,R. S., et al. Nat Med 9, 343-346 (2003); Hessell, A. J., et al. PLoSPathog 5, e1000433 (2009); Parren, P. W., et al. J Virol 75, 8340-8347(2001); Mascola, J. R. Vaccine 20, 1922-1925 (2002); Mascola, J. R., etal. Nat Med 6, 207-210 (2000); Mascola, J. R., et al. J Virol 73,4009-4018 (1999)). Therefore, broadly neutralizing antibodies (bNAbs)are considered to be the types of antibodies that should be elicited bya vaccine. Unfortunately, existing immunogens, often designed based onthese bNAbs, have failed to elicit NAb responses of the required breadthand potency. Therefore, it is of high priority to identify new bNAbsthat bind to epitopes that may be more amenable to incorporation intoimmunogens for elicitation of NAb responses.

The present invention provides a novel method for isolating novel broadand potent neutralizing monoclonal antibodies against HIV. The methodinvolves selection of a PBMC donor with high neutralization titer ofantibodies in the plasma. B cells are screened for neutralizationactivity prior to rescue of antibodies. Novel broadly neutralizingantibodies are obtained by emphasizing neutralization as the initialscreen.

The invention relates to potent, broadly neutralizing antibody (bNAb)wherein the antibody neutralizes HIV-1 species belonging to two or moreclades, and further wherein the potency of neutralization of at leastone member of each clade is determined by an IC50 value of less than 0.2μg/mL. In some aspects, the clades are selected from Clade A, Clade B,Clade C, Clade D and Clade AE. In some aspects, the HIV-1 belonging twoor more clades are non-Clade B viruses. In some aspects, the broadlyneutralizing antibody neutralizes at least 60% of the HIV-1 strainslisted in Tables 18A-18F. In some embodiments, at least 70%, or at least80%, or at least 90% of the HIV-1 strains listed in Tables 18A-18F areneutralized.

The invention relates to potent, broadly neutralizing antibody (bNAb)wherein the antibody neutralizes HIV-1 species with a potency ofneutralization of at least a plurality of HIV-1 species with an IC50value of less than 0.2 μg/mL. In some embodiments the potency ofneutralization of the HIV-1 species has an IC50 value of less than 0.15μg/mL, or less than 0.10 μg/mL, or less than 0.05 μg/mL. In someaspects, a potent, broadly neutralizing antibody is defined as a bNAbthat displays a potency of neutralization of at least a plurality ofHIV-1 species with an IC90 value of less than 2.0 μg/mL. In someembodiments the potency of neutralization of the HIV-1 species has anIC90 value of less than 1.0 μg/mL, or less than 0.5 μg/mL.

An exemplary method is illustrated in the schematic shown in FIG. 4.Peripheral Blood Mononuclear Cells (PBMCs) were obtained from anHIV-infected donor selected for HIV-1 neutralizing activity in theplasma. Memory B cells were isolated and B cell culture supernatantswere subjected to a primary screen of neutralization assay in a highthroughput format. Optionally, HIV antigen binding assays using ELISA orlike methods were also used as a screen. B cell lysates corresponding tosupernatants exhibiting neutralizing activity were selected for rescueof monoclonal antibodies by standard recombinant methods.

In one embodiment, the recombinant rescue of the monoclonal antibodiesinvolves use of a B cell culture system as described in Weitcamp J-H etal., J. Immunol. 171:4680-4688 (2003). Any other method for rescue ofsingle B cells clones known in the art also may be employed such as EBVimmortalization of B cells (Traggiai E., et al., Nat. Med. 10(8):871-875(2004)), electrofusion (Buchacher, A., et al., 1994. AIDS Res. Hum.Retroviruses 10:359-369), and B cell hybridoma (Karpas A. et al., Proc.Natl. Acad. Sci. USA 98:1799-1804 (2001).

In some embodiments, monoclonal antibodies were rescued from the B cellcultures using variable chain gene-specific RT-PCR, and transfectantwith combinations of H and L chain clones were screened again forneutralization and HIV antigen binding activities. mAbs withneutralization properties were selected for further characterization.

A novel high-throughput strategy was used to screen IgG-containingculture screening supernatants from approximately 30,000 activatedmemory B cells from a clade A infected donor for recombinant, monomericgp120JR-CSF and gp41HxB2 (Env) binding as well as neutralizationactivity against HIV-1JR-CSF and HIV-1SF162 (See Table 1).

TABLE 1 Memory B ceil Screening. Total number of wells screened 23,328Number of sIgG⁺memory B cells screened 30,300 gp120 ELISA hits 411(1.36%) gp41 ELISA hits 167 (0.55%) SF162 neutralization hits 401(1.32%) JR-CSF neutralization hits 401 (1.32%)

Unexpectedly, a large proportion of the B cell supernatants thatneutralized HIV-1JR-CSF did not bind monomeric gp120JR-CSF or gp41HxB2,and there were only a limited number of cultures that neutralized bothviruses (FIG. 3B). Antibody genes were rescued from five B cell culturesselected for differing functional profiles; one bound to gp120 and onlyneutralized HIV-1SF162, two bound to gp120 and weakly neutralized bothviruses, and two potently neutralized HIV-1JR-CSF, failed to neutralizeHIV-1SF162, and did not bind to monomeric gp120 or gp41. Five antibodiesidentified according to these methods are disclosed herein. Theantibodies were isolated from a human sample obtained throughInternational AIDS Vaccine Initiative's (IAVI's) Protocol G, and areproduced by the B cell cultures referred to as 1443_C16, 1456_P20,1460_G14, 1495_C14 or 1496_C09. Antibodies referred to as 1443_C16(PG16), 1456_P20 (PG20), 1460_G14 (PGG14), 1495_C14 (PGC14) or 1496_C09(PG9), were isolated from the corresponding B cell cultures. Theseantibodies have been shown to neutralize HIV in vitro. Analysis of theantibody variable genes revealed that two antibody pairs were related bysomatic hypermutation and that two of the somatic variants containedunusually long CDRH3 loops (Table 2). Long CDRH3 loops have previouslybeen associated with polyreactivity. (Ichiyoshi, Y. & Casali, P. J ExpMed 180, 885-895 (1994)). The antibodies were tested against a panel ofantigens and the antibodies were confirmed to be not polyreactive.

TABLE 2 Sequence Analysis of mAb Variable Genes SEQ SEQ GermlineGermline ID ID Clone IGVL^(a) IGVH^(a) CDRL3^(b) NO: CDRH3^(b) NO: PG16VL2- VH3- SSLTDRSHRIF 1 EAGGPIWHDDVKYYDFNDGYYNYHYM 6 14*01 33*05 DV PG9VL2- VH3- KSLTSTRRRVF 2 EAGGPDYRNGYNYYDFYDGYYNYHYM 7 14*01 33*05 DVPGG14 VK1- VH1- SYSTPRTF 3 DRRVVPMATDNWLDP 8 39*01 69*12 PG20 VK2- VH1-SFSTPRTF 4 DRRAVPIATDNWLDP 9 14*01 69*12 PGC14 VL3- VH1- AWETTTTTFVFF 5GAVGADSGSWFDP 10 1*01 24*01 ^(a)Germ line gene sequences were determinedusing the IMGT database, which is publicly available at imgt.cines.fr.“L” and “K” refer to lamda and kappa chains, respectively, ^(b)Boldedamino acids denote differences between somatic variants.

TABLE 3A Heavy Chain Gene Usage Summary V- mAb Gene & V-Gene J-GeneJ-Gene mAb ID Specificity allele identity & allele identity CDR31443_C16 ELISA- IGHV3- 0.8507 IGHJ6*03 0.8548AREAGGPIWHDDVKYYDFNDGYYNYHYMDV negative 33*05 (245/288 (53/62 (SEQ IDNO: 46) nt) nt) 1456_P20 gp120 IGHV1- 0.8507 IGHJ5*02 0.8824ARDRRAVPIATDNWLDP (SEQ ID NO: 47) 69*11 (245/288 (45/51 or nt) nt)IGHV1- 69*12 1460_G14 gp120 IGHV1- 0.8611 IGHJ5*02 0.8627TRDRRVVPMATDNWLDP (SEQ ID NO: 48) 69*11 (248/288 (44/51 or nt) nt)IGHV1- 69*12 1495_C14 gp120 IGHV1- 0.8889 IGHJ5*02 0.8431AAGAVGADSGSWFDP (SEQ ID NO: 49) f*01 (256/288 (43/51 nt) nt) 1496_C09ELISA- IGHV3- 0.8507 IGHJ6*03 0.8387 VREAGGPDYRNGYNYYDFYDGYYNYHYMDVnegative 33*05 (245/288 (52/62 (SEQ ID NO: 50) nt) nt)

TABLE 3B Light Chain Gene Usage Summary SEQ mAb V-Gene V-gene J-GENEJ-Gene ID mAb ID Specificity and allele identity and allele identityCDR3 NO: 1443_C16 ELISA- IGLV2- 0.8819 IGLJ2*01. 0.8333 SSLTDRSHRI 41negative 14*01 (254/288 or (30/36 nt) nt) IGLJ3*01 or IGLJ3*02 1456_P20gp120 IGKV1- 0.9211 IGKJ5*01 0.9211 QQSFSTPRT 42 39*01, or (257/279(35/38 nt) IGKV1D- nt) 39*01 1460_G14 gp120 IGKV1- 0.9211 IGKJ5*010.8947 QQSYSTPRT 43 39*01, or (257/279 (34/38 nt) IGKV1D- nt) 39*011495_C14 gp120 IGLV3- 0.8889 IGLJ2*01. 0.8684 QAWETTTTTFVF 44 1*01(248/279 or (33/38 nt) nt) IGLJ3*01 1496_C09 ELISA- IGLV2- 0.9132IGLJ3*02 0.8611 KSLTSTRRRV 45 negative 14*01 (263/288 (31/36 nt) nt)

The broadly neutralizing antibodies from 1443_C16 (PG16) and 1496_C09(PG9) clones obtained by this method did not exhibit soluble gp120 orgp41 binding at levels that correlate with neutralization activity. Themethod of the invention therefore allows identification of novelantibodies with broad cross-clade neutralization properties regardlessof binding activities in an ELISA screen. Further characterization ofPG16 and PG9 is disclosed herein.

All five antibodies were first tested for neutralization activityagainst a multi-clade 16-pseudovirus panel (Table 4). Two of theantibodies that bound to monomeric gp120 in the initial screen (PGG14and PG20) did not show substantial neutralization breadth or potencyagainst any of the viruses tested, and the third antibody that bound togp120 (PGC14) neutralized 4/16 viruses with varying degrees of potency.In contrast, the two antibodies that failed to bind recombinant Env inthe initial screen (PG9 and PG16) neutralized a large proportion of theviruses at sub-microgram per ml concentrations. PG9 and PG16 neutralizednon-clade B viruses with greater breadth than three out of the fourexisting bNAbs. This is significant considering that the majority ofHIV-1 infected individuals worldwide are infected with non-clade Bviruses.

TABLE 4 Neutralization Profiles of Rescued mAbs

^(a)Plateau observed in curve.

Table 17A shows neutralization profiles (IC50 values) of monoclonalantibodies 1443_C16 (PG16), 1456_P20 (PG20), 1460_G14 (PGG14), 1495_C14(PGC14) and 1496_C09 (PG9) and the known cross-clade neutralizingantibodies b12, 2G12, 2F5 and 4E10 on a diverse panel of 16 HIVpseudoviruses from different clades. 1443_C16 (PG16) and 1496_C09 (PG9)neutralize HIV-1 species from Clades A, B, C, D and CRF01_AE with betterpotency for most viral strains tested than known and generally acceptedbroad and potent neutralizing antibodies. However, neutralizationprofiles of individual species of HIV-1 belonging to these clades varybetween 1443_C16 (PG16) and 1496_C09 (PG9) and the known cross-cladeneutralizing antibodies b12, 2G12, 2F5 and 4E10. 1495_C14 (PGC14)neutralizes fewer HIV-1 species from Clades A, B and C comparable toother neutralizing antibodies. Table 17B shows IC90 values of themonoclonal antibodies 1443_C16 (PG16) and 1496_C09 (PG9) and the knowncross-clade neutralizing antibodies b12, 2G12, 2F5 and 4E10 on the samepanel of pseudoviruses. FIG. 4 shows neutralization activities ofmonoclonal antibodies 1443_C16 (PG16) and 1496_C09 (PG9) to six otherHIV pseudoviruses (YU2, Ba1, ADA, DU172, DU422, and ZM197) for clades Band C not included in Tables 17A and 17B.

PG9, PG16, and PGC14 were next evaluated on a large multi-cladepseudovirus panel consisting of 162 viruses to further assess theneutralization breadth and potency of these three antibodies (Tables5A-5B, Tables 18A-18F and Tables 19A-19B). The bNAbs b12, 2G12, 2F5, and4E10, as well as the donor's serum, were also included in the panel forcomparison. Overall, PG9 neutralized 127 out of 162 and PG16 neutralized119 out of 162 viruses with a potency that frequently considerablyexceeded that noted for the four control bNAbs.

The median IC50 and IC90 values for neutralized viruses across allclades were an order of magnitude lower for PG9 and PG16 than any of thefour existing bNAbs (Table 5A, Tables 18A-18F and Tables 19A-19B). BothmAbs showed overall greater neutralization breadth than b12, 2G12, and2F5 (Table 5B, Tables 18A-18F and Tables 19A-19B). At low antibodyconcentrations, PG9 and PG16 also demonstrated greater neutralizationbreadth than 4E10 (Table 5B). Furthermore, both mAbs potentlyneutralized one virus (IAVI-C18) that exhibits resistance to all fourexisting bNAbs (Tables 18A-18F). The mAb neutralization curves revealthat, whereas the PG9 neutralization curves usually exhibit sharpslopes, the neutralization curves for PG16 sometimes exhibit gradualslopes or plateaus at less than 100% neutralization. Althoughneutralization curves with similar profiles have been reportedpreviously (W. J. Honnen et al., J Virol 81, 1424 (February, 2007), A.Pinter et al., J Virol 79, 6909 (June, 2005)), the mechanism for this isnot well understood.

Comparison of the neutralization profile of the serum with theneutralization profile of PG9, PG16 and PGC14 revealed that these threeantibodies could recapitulate the breadth of the serum neutralization inmost cases (Tables 18A-18F). For example, almost all of the viruses thatwere neutralized by the serum with an IC50>1:500 were neutralized by PG9and/or PG16 at <0.05 μg/mL. The one case where this did not occur wasagainst HIV-1SF162, but this virus was potently neutralized by PGC14.Despite the fact that PG9 and PG16 are somatic variants, they exhibiteddifferent degrees of potency against a number of the viruses tested. Forinstance, PG9 neutralized HIV-16535.30 approximately 185 times morepotently than PG16, and PG16 neutralized HIV-1MGRM-AG-001 approximately440 times more potently than PG9. In some cases, the two antibodies alsodiffered in neutralization breadth; PG9 neutralized nine viruses thatwere not affected by PG16, and PG16 neutralized two viruses that werenot affected by PG9. Based on these results, it is postulated that broadserum neutralization might be mediated by somatic antibody variants thatrecognize slightly different epitopes and display varying degrees ofneutralization breadth and potency against any given virus. In the faceof an evolving viral response, it seems reasonable that the immunesystem might select for these types of antibodies.

Comparison of the neutralization profile of the serum with theneutralization profile of PG9, PG16 and PGC14 revealed that these threeantibodies could recapitulate the breadth of the serum neutralization inmost cases. For example, almost all of the viruses that were neutralizedby the serum with an IC50>1:1000 were neutralized by PG9 and/or PG16 at<0.005 μg/mL. The one case where this did not occur was againstHIV-1SF162, but this virus was potently neutralized by PGC14. Tables5(a) and 5(b) show the neutralization activities—breadth and potency,respectively—of PG9, PG16, and PGC14 as well as four control bNAbs asmeasured by IC50 values. Tables 19A-19B show results of the sameanalysis using IC₉₀ values.

TABLE 5(A) Neutralization Potency of mAbs

Boxes are color coded as follows: white, median potency >50 μg/mL:,light grey, median potency between 2 and 20 μg/mL; medium grey, medianpotency between 0.2 and 2 μg/mL; dark grey, median potency <0.2 μg/mL.^(a)CRF_07BC and CRF_08BC viruses are not included in the clade analysisbecause there was only one virus tested from each of these clades.

TABLE 5(B) Neutralization Breadth of mAbs

Boxes are color coded as follows: white, no viruses neutralized; black,1 to 30% of viruses neutralized; light grey, 30 to 60% of virusesneutralized; medium grey, 60 to 90% of viruses neutralized; dark grey,90 to 100% of viruses neutralized. ^(a)CRF_07BC and CRF_08BC viruses arenot included in the clade analysis because there was only one virustested from each of these clades.

Despite the fact that PG9 and PG6 are somatic variants, they exhibiteddifferent degrees of potency against a number of the viruses tested. Forinstance, PG9 neutralized the virus 6535.30 about 100 times morepotently than PG16, and PG16 neutralized the virus MGRM-AG-001 about3000 times more potently than PG9. In some cases, the two antibodiesalso differed in neutralization breadth; PG9 neutralized seven virusesthat were not neutralized by PG16, and PG16 neutralized three virusesthat were not neutralized by PG9. Without being bound by theory, itappears that broad serum neutralization might be mediated by somaticvariants that recognize slightly different epitopes and display varyingdegrees of neutralization breadth and potency against any given virus.In the face of an evolving viral response, the immune system likelyselects for these types of antibodies.

The antibodies were also tested for ability to bind soluble recombinantHIV envelope proteins. FIG. 5 shows dose response curves of 1456_P20(PG20), 1495_C14 (PGC14) and 1460_G14 (PGG4) binding to recombinantgp120 in ELISA as compared to control anti-gp120 (b12). FIG. 6 showsELISA binding assays of monoclonal antibodies 1443_C16 (PG16) and1496_C09 (PG9) to HIV-1 strain YU2 gp140 and JR-CSF gp120, the membraneproximal region (MPER) of HIV-1 envelope glycoprotein gp41, and the V3polypeptide. PG-9 binds to YU2 gp140 (IC₅₀ ˜20-40 nM), YU2 gp120 andweakly binds to JR-CSF gp20. However, PG16 weakly binds Yu2 gp120, butnot the soluble form of HIV-1 envelope glycoprotein, gp120 JR-CSF.Neither mAb binds to JR-FL gp120, JR-FL gp40, MPER peptide of gp41 or V3peptide.

FIG. 7 shows binding of monoclonal antibodies 1443_C16 (PG16) and1496_C09 (PG9) to HIV-1 YU2 gp160 expressed on the cell surface in thepresence and absence of sCD4. Competitive inhibition of the binding bysCD4 indicates that the binding of monoclonal antibody 1496_C09 to HIV-1envelope protein gp160 expressed on the cell surface is presumablyaffected due to the conformational changes induced by sCD4. The datafurther suggest that 1443_C16 (PG16) and 1496_C09 (PG9) exhibitrelatively stronger binding to trimeric forms of the HIV-1 Env (gp160and gp40) than to the monomeric gp120.

FIG. 8 shows binding of monoclonal antibodies 1443_C16 (PG16) and1496_C09 (PG9) to HIV-1 transfected cells. PG9 and PG16 do not binduntransfected cells. PG9 and PG16 bind JR-CSF, ADA, and YU2 gp160transfected cells. PG9 and PG16 do not bind JR-FL gp160 transfectedcells (cleaved or uncleaved). PG9 and PG16 do not bind ADA ΔV1/ΔV2transfected cells. PG9 and PG16 binding to JR-CSF gp160 transfectedcells is inhibited by sCD4.

FIG. 9 shows the capturing of entry-competent JR-CSF pseudovirus byneutralizing monoclonal antibodies 1443_C16 (PG16) and 1496_C09 (PG9) ina dose-dependent manner. The ability of both antibodies to captureJR-CSF pseudovirus is higher than IgG b12 but comparable to IgG2G12. Itis postulated that the capture may be mediated by the binding of themAbs to the HIV-1 Env on the virions.

FIG. 10A shows that sCD4, PG16 and PG9 compete for the binding ofmonoclonal antibody 1443_C16 (PG16) to JR-CSF pseudovirus but b12, 2G12,2F5 and 4E10 do not. FIG. 10B shows sCD4. PG16 and PG9 compete for thebinding of monoclonal antibody 1496_C09 (PG9) to JR-CSF pseudovirus butb12, 2G12, 2F5 and 4E10 do not. This suggests that the PG16 and PG9 mAbsbind gp120 at a site different from those bound by b12 and 2G12. PG9 andPG16 binding to HIV-1 envelope protein is competitively inhibited bysCD4. Given that the MAbs are not inhibited by the CD4 binding site MAbb12, this suggests that PG9 and PG16 are binding to an epitope that isunavailable for sCD4 binding to gp120 as a result of conformationalchanges. The inability of PG9 and PG16 to bind monomeric gp120JR-CSF orgp41HxB2 in the initial screen while potently neutralizing HIV-1JR-CSFsuggests that the epitope targeted by these antibodies is preferentiallyexpressed on trimeric HIV envelope protein. The ability of PG9 and PG16to bind monomeric gp120 from several different strains, artificiallytrimerized gp140 constructs, and trimeric Env expressed on the surfaceof transfected cells respectively, was compared. Although bothantibodies bound with high affinity to cell surface Env, PG16 did notbind to any of the soluble gp120 or gp140 constructs and PG9 bound onlyweakly to monomeric gp120 and trimerized gp140 from certain strains(FIG. 11). It has been previously shown that a substantial fraction ofcell surface Env is comprised of uncleaved gp160 molecules. (Pancera, M.& Wyatt, R. Virology 332, 145-156 (2005)). That PG9 and PG16 do notexhibit exclusive specificity for native HIV-1 trimers was confirmed bythe fact that both antibodies bound with high affinity tocleavage-defective HIV-1YU2 trimers expressed on the surface oftransfected cells (FIG. 12).

The epitopes recognized by PG9 and PG16 were investigated. Since the PG9and PG16 antibodies are somatic variants, they recognize the same oroverlapping epitopes. Both antibodies cross-competed for binding toHIV-1JR-CSF transfected cells (FIG. 13A). Ligation of monomeric gp120 orcell surface Env with soluble CD4 diminished binding of both PG9 andPG16, although neither antibody competed with CD4-binding siteantibodies for trimer binding (FIG. 13A-13C). This result suggests thatCD4-induced conformational changes cause a loss of the epitope targetedby the antibodies.

Since PG9 bound well enough to gp120 from certain isolates to generateELISA binding curves, competition ELISAs were performed with PG9 using apanel of neutralizing and non-neutralizing antibodies. These datarevealed that PG9 cross-competed with anti-V2, anti-V3, and to a lesserextent, CD4i antibodies for gp120. (FIGS. 13D and 14).

Neither PG9 nor PG16 bound to V1/V2 or V3 deleted HIV-1JR-CSF variantsexpressed on the surface of transfected cells, further suggestingcontributions of variable loops in forming their epitopes (FIG. 13E).

To dissect the fine specificity of PG9 and PG16, alanine scanning wasperformed using a large panel of HIV-1JR-CSF Env alanine mutants thathave been described previously (Pantophlet, R., et al. J Virol 77,642-658 (2003); Pantophlet, R., et al. J Virol 83, 1649-1659 (2009);Darbha, R., et al. Biochemistry 43, 1410-1417 (2004); Scanlan, C. N., etal. J Virol 76, 7306-7321 (2002)) as well as several new alaninemutants. Pseudoviruses incorporating single Env alanine mutations weregenerated, and PG9 and PG16 were tested for neutralization activityagainst each mutant pseudovirus. Mutations that resulted in viral escapefrom PG9 and PG16 neutralization were considered important for formationof the PG9 and PG16 epitopes (Tables 12 and 13).

Based on this criteria, and consistent with the competition experiments,residues that form the epitopes recognized by PG9 and PG16 appear to belocated in conserved regions of the V2 and V3 loops of gp120. Certainco-receptor binding site mutations also had an effect on PG9 and PG16neutralization, albeit to a lesser extent. Generally, PG9 and PG16 weredependent on the same residues, although PG16 was more sensitive tomutations located in the tip of the V3 loop than PG9. Interestingly,although neither antibody bound to wild-type HIV-1JR-FL transfectedcells, a D to K mutation at position 168 in the V2 loop of HIV-1JR-FLgenerated high-affinity PG9 and PG16 recognition (Tables 18A-18F). N156and N160, sites of V2 N-glycosylation, also appear to be critical informing the epitope since substitutions at these positions resulted inescape from PG9 and PG16 neutralization. Deglycosylation of gp120abolished binding of PG9 (FIG. 16), confirming that certain glycans maybe important in forming the epitope.

HIV-1 SF162 contains a rare N to K polymorphism at position 160, andmutation of this residue to an Asn renders this isolate sensitive to PG9and PG16 (FIG. 17).

The preferential binding of PG9 and PG16 to native trimers could eitherbe a consequence of gp120 subunit cross-linking or recognition of apreferred oligomeric gp120 conformation. To address this question, thebinding profiles of PG9 and PG16 to mixed HIV-1YU2 trimers wereexamined, in which two gp120 subunits containing point mutationsabolished binding of the two antibodies. A third substitution thatabrogates binding of 2G12, which binds with high affinity to bothmonomeric gp120 and trimeric Env, was also introduced into the sameconstruct as an internal control. Cell surface binding analysis revealedthat all three antibodies bound to the mixed trimers with similarapparent affinity as to wild-type trimers and all saturated at a similarlower level (FIG. 18). This result suggests that the preference of PG9and PG16 for trimeric Env is due to gp120 subunit presentation in thecontext of the trimeric spike rather than gp120 cross-linking.

It has been shown that NAbs that bind to epitopes encompassing parts ofthe V2 or both the V2 and V3 domains can exhibit potency comparable tothat of PG9 and PG16, although these antibodies have thus far displayedstrong strain-specificity. (Honnen, W. J., et al. J Virol 81, 1424-1432(2007); Gorny, M. K., et al. J Virol 79, 5232-5237 (2005)). Importantly,the epitopes recognized by these antibodies have been shown to differfrom that of the clade B consensus sequence only by single amino acidsubstitutions, which suggested the existence of a relatively conservedstructure within the V2 domain. (Honnen, W. J., et al. J Virol 81,1424-1432 (2007)). The results observed with PG9 and PG16 confirm thatthis region serves as a potent neutralization target and demonstratesthat antibodies that recognize conserved parts of V2 and V3 can possessbroad reactivity.

The invention is based on novel monoclonal antibodies and antibodyfragments that broadly and potently neutralize HIV infection. In someembodiments, these monoclonal antibodies and antibody fragments have aparticularly high potency in neutralizing HIV infection in vitro acrossmultiple clades or across a large number of different HIV species. Suchantibodies are desirable, as only low concentrations are required toneutralize a given amount of virus. This facilitates higher levels ofprotection while administering lower amounts of antibody. Humanmonoclonal antibodies and the immortalized B cell clones that secretesuch antibodies are included within the scope of the invention.

The invention provides methods for using high throughput functionalscreening to select neutralizing antibodies with unprecedented breadthand potency. The invention relates to other potent, and broadlyneutralizing antibodies that can be developed using the same methods. Inparticular, the invention relates to potent, broadly neutralizingantibodies against different strains of HIV, wherein the bNAbs bindpoorly to recombinant forms of Env. The invention provides twoneutralizing antibodies, PG9 and PG16, with broad neutralizingactivities particularly against non-clade B isolates. The inventionprovides vaccine-induced antibodies of high specificity that provideprotection against a diverse range of the most prevalent isolates of HIVcirculating worldwide. The invention provides antibodies with very highand broad neutralization potency, such as that exhibited by PG9 and PG16in vitro, which provides protection at relatively modest serumconcentrations, and are generated by vaccination unlike the broad NAbsknown in the art. The invention provides immunogens that can be designedthat focus the immune response on conserved regions of variable loops inthe context of the trimeric spike of the gp120 subunit of the Envprotein.

The invention also relates to the characterization of the epitope towhich the antibodies bind and the use of that epitope in raising animmune response.

The invention also relates to various methods and uses involving theantibodies of the invention and the epitopes to which they bind. Forexample, monoclonal antibodies according to the invention can be used astherapeutics. In some aspects, the monoclonal antibodies are used foradjuvant therapy. Adjuvant therapy refers to treatment with thetherapeutic monoclonal antibodies, wherein the adjuvant therapy isadministered after the primary treatment to increase the chances of acure or reduce the statistical risk of relapse.

The invention provides novel monoclonal or recombinant antibodies havingparticularly high potency in neutralizing HIV. The invention alsoprovides fragments of these recombinant or monoclonal antibodies,particularly fragments that retain the antigen-binding activity of theantibodies, for example which retain at least one complementaritydetermining region (CDR) specific for HIV proteins. In thisspecification, by “high potency in neutralizing HIV” is meant that anantibody molecule of the invention neutralizes HIV in a standard assayat a concentration lower than antibodies known in the art.

Preferably, the antibody molecule of the present invention canneutralize at a concentration of 0.16 μg/ml or lower (i.e. 0.15, 0.125,0.1, 0.075, 0.05, 0.025, 0.02, 0.016, 0.015, 0.0125, 0.01, 0.0075,0.005, 0.004 or lower), preferably 0.016 μg/ml or lower (an antibodyconcentration of 10⁻⁸ or lower, preferably 10⁻⁹ M or lower, preferably10⁻¹⁰ M or lower, i.e. 10⁻¹¹ M, 10⁻¹² M, 10⁻¹³ M or lower). This meansthat only very low concentrations of antibody are required for 50%neutralization of a clinical isolate of HIV in vitro. Potency can bemeasured using a standard neutralization assay as described in the art.

The antibodies of the invention are able to neutralize HIV. Monoclonalantibodies can be produced by known procedures, e.g., as described by R.Kennet et al. in “Monoclonal Antibodies and Functional Cell Lines;Progress and Applications”. Plenum Press (New York), 1984. Furthermaterials and methods applied are based on known procedures, e.g., suchas described in J. Virol. 67:6642-6647, 1993.

These antibodies can be used as prophylactic or therapeutic agents uponappropriate formulation, or as a diagnostic tool.

A “neutralizing antibody” is one that can neutralize the ability of thatpathogen to initiate and/or perpetuate an infection in a host and/or intarget cells in vitro. The invention provides a neutralizing monoclonalhuman antibody, wherein the antibody recognizes an antigen from HIV.

Preferably an antibody according to the invention is a novel monoclonalantibody referred to herein as 1496_C09 (PG9), 1443_C16 (PG16), 1456_P20(PG20), 1460_G14 (PGG14), and 1495_C14 (PGC14). These antibodies wereinitially isolated from human samples and are produced by the B cellcultures referred to as 1443_C16, 1456_P20, 1460_G14, 1495_C14 or1496_C09. These antibodies have been shown to neutralize HIV in vitro.PG9 and PG16 have been shown to have broad, potent HIV neutralizingactivity.

The CDRs of the antibody heavy chains are referred to as CDRH1, CDRH2and CDRH3, respectively. Similarly, the CDRs of the antibody lightchains are referred to as CDRL1, CDRL2 and CDRL3, respectively. Theposition of the CDR amino acids are defined according to the IMGTnumbering system as: CDR1--IMGT positions 27 to 38, CDR2--IMGT positions56 to 65 and CDR3--IMGT positions 105 to 117. (Lefranc, M P. et al. 2003IMGT unique numbering for immunoglobulin and T cell receptor variableregions and Ig superfamily V-like domains. Dev Comp Immunol.27(1):55-77; Lefranc, M P. 1997. Unique database numbering system forimmunogenetic analysis. Immunology Today, 18:509; Lefranc, M P. 1999.The IMGT unique numbering for Immunoglobulins, T cell receptors andIg-like domains. The Immunologist, 7:132-136.)

The amino acid sequences of the CDR3 regions of the light and heavychains of the antibodies are shown in Tables 3A and 3B.

A phylogram is a branching diagram (tree) assumed to be an estimate ofphylogeny, branch lengths are proportional to the amount of inferredevolutionary change. Tree diagrams of the five heavy chains and the fivelight chains were prepared using ClustalW (Larkin M. A., BlackshieldsG., Brown N. P., Chenna R., McGettigan P. A., McWilliam H., Valentin F.,Wallace I. M., Wilm A., Lopez R., Thompson J. D., Gibson T. J. andHiggins D. G. Bioinformatics 23(21): 2947-2948 (2007); Higgins D G etal. Nucleic Acids Research 22: 4673-4680. (1994)) and are shown in FIGS.1A and 1B respectively.

The sequences of the antibodies were determined, including the sequencesof the variable regions of the Gamma heavy and Kappa or Lambda lightchains of the antibodies designated 1496_C09 (PG9), 1443_C16 (PG16),1456_P20 (PG20), 1460_G14 (PGG14), and 1495_C14 (PGC14). In addition,the sequence of each of the polynucleotides encoding the antibodysequences was determined. Shown below are the polypeptide andpolynucleotide sequences of the gamma heavy chains and kappa lightchains, with the signal peptides at the N-terminus (or 5′ end) and theconstant regions at the C-terminus (or 3′ end) of the variable regions,which are shown in bolded text.

1443_C16 (PG16) gamma heavy chain nucleotide sequence: 1443 C16 γ3coding sequence (variable region in bold)

(SEQ ID NO: 11) ATGGAGTTTGGGCTGAGCTGGGTTTTCCTCGCAACTCTGTTAAGAGTTGTGAAGTGTCAGGAACAACTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCGGGGGGGTCCCTGAGACTCTCCTGTTTAGCGTCTGGATTCACGTTTCACAAATATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGCCTGGAGTGGGTGGCACTCATCTCAGATGACGGAATGAGGAAATATCATTCAGACTCCATGTGGGGCCGAGTCACCATCTCCAGAGACAATTCCAAGAACACTCTTTATCTGCAATTCAGCAGCCTGAAAGTCGAAGACACGGCTATGTTCTTCTGTGCGAGAGAGGCTGGTGGGCCAATCTGGCATGACGACGTCAAATATTACGATTTTAATGACGGCTACTACAACTACCACTACATGGACGTCTGGGGCAAGGGGACCACGGTCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTC CGGGTAAATGA

1443_C16 (PG16) gamma heavy chain variable region nucleotide sequence:

(SEQ ID NO: 99) CAGGAACAACTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCGGGGGGGTCCCTGAGACTCTCCTGTTTAGCGTCTGGATTCACGTTTCACAAATATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGCCTGGAGTGGGTGGCACTCATCTCAGATGACGGAATGAGGAAATATCATTCAGACTCCATGTGGGGCCGAGTCACCATCTCCAGAGACAATTCCAAGAACACTCTTTATCTGCAATTCAGCAGCCTGAAAGTCGAAGACACGGCTATGTTCTTCTGTGCGAGAGAGGCTGGTGGGCCAATCTGGCATGACGACGTCAAATATTACGATTTTAATGACGGCTACTACAACTACCACTACATGGACGTCTGGGGCAAGGGGACCACGGTCA CCGTCTCGAGC

1443_C16 (PG16) gamma heavy chain amino acid sequence: expressed proteinwith variable region in bold.

(SEQ ID NO: 12) QEQLVESGGGVVQPGGSLRLSCLASGFTFHKYGMHWVRQAPGKGLEWVALISDDGMRKYHSDSMWGRVTISRDNSKNTLYLQFSSLKVEDTAMFFCAREAGGPIWHDDVKYYDFNDGYYNYHYMDVWGKGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK

1443_C16 (PG16) gamma heavy chain variable region amino acid sequence:(Kabat CDRs underlined, Chothia CDRs in bold italics)

(SEQ ID NO: 31) QEQLVESGGGVVQPGGSLRLSCLA

WVRQAPGKGLEWVA

GRVTISR DNSKNTLYLQFSSLKVEDTAMFFCAR

WGKGTTVTVSS

1443_C16 (PG16) gamma heavy chain Kabat CDRs:

CDR 1: (SEQ ID NO: 88) SGFTFHKYGMH CDR 2: (SEQ ID NO: 89)LISDDGMRKYHSDSMW CDR 3: (SEQ ID NO: 6) EAGGPIWHDDVKYYDFNDGYYNYHYMDV

1443_C16 (PG16) gamma heavy chain Chothia CDRs:

CDR 1: (SEQ ID NO: 88) SGFTFHKYGMH CDR 2: (SEQ ID NO: 89)LISDDGMRKYHSDSMW CDR 3: (SEQ ID NO: 6) EAGGPIWHDDVKYYDFNDGYYNYHYMDV

1443_C16 (PG16) lambda light chain nucleotide sequence: 1443_C16 λ2coding sequence (variable region in bold)

(SEQ ID NO: 13) ATGGCCTGGGCTCTGCTATTCCTCACCCTCTTCACTCAGGGCACAGGGTCCTGGGGCCAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGACGATCACCATCTCCTGCAATGGAACCAGCAGTGACGTTGGTGGATTTGACTCTGTCTCCTGGTACCAACAATCCCCAGGGAAAGCCCCCAAAGTCATfGGTTTTTGATGTCAGTCATCGGCCCTCAGGTATCTCTAATCGCTTCTCTGGCTCCAAGTCCGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCACATTGAGGACGAGGGCGATTATTTCTGCTCTTCACTGACAGACAGAAGCCATCGCATATTCGGCGGCGGGACCAAGGTGACCGTTCTAGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTACCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAAAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAA TGTTCATAG

1443_C16 (PG16) lambda light chain variable region nucleotide sequence:

(SEQ ID NO: 100) CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGACGATCACCATCTCCTGCAATGGAACCAGCAGTGACGTTGGTGGATTTGACTCTGTCTCCTGGTACCAACAATCCCCAGGGAAAGCCCCCAAAGTCATGGTTTTTGATGTCAGTCATCGGCCCTCAGGTATCTCTAATCGCTTCTCTGGCTCCAAGTCCGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCACATTGAGGACGAGGGCGATTATTTCTGCTCTTCACTGACAGACAGAAGCCATCGCATATTCGGCGGCGGGACCAAGGTGACCGTTCTA

1443_C16 (PG16) lambda light chain amino acid sequence: expressedprotein with variable region in bold.

(SEQ ID NO: 14) QSALTQPASVSGSPGQTITISCNGTSSDVGGFDSVSWYQQSPGKAPKVMVFDVSHRPSGISNRFSGSKSGNTASLTISGLHIEDEGDYFCSSLTDRSHRIFGGGTKVTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVT HEGSTVEKTVAPTECS

1443_C16 (PG16) lambda light chain variable region amino acid sequence:(Kabat CDRs underlined, Chothia CDRs in bold italics)

(SEQ ID NO: 32) QSALTQPASVSGSPGQTITISC

WYQQSPGKAPKV MVF

ISNRFSGSKSGNTASLTISGLHIEDEGDYFC

FGGGTKVTVL

1443_C16 PG16 lambda light chain Kabat CDRs:

CDR 1: NGTSSDVGGFDSVS (SEQ ID NO: 97) CDR 2: DVSHRPSG (SEQ ID NO: 95)CDR 3: SSLTDRSHRI (SEQ ID NO: 41)

1443_C16 (PG16) lambda light chain Chothia CDRs:

CDR 1: NGTSSDVGGFDSVS (SEQ ID NO: 97) CDR 2: DVSHRPSG (SEQ ID NO: 95)CDR 3: SSLTDRSHRI (SEQ ID NO: 41)

1456_P20 (PG20) gamma heavy chain nucleotide sequence: 1456_P20 γ1coding sequence (variable region in bold)

(SEQ ID NO: 15) ATGGACTGGATTTGGAGGTTCCTCTTTGTGGTGGCAGCAGCTACAGGTGTCCAGTCCCAGGTCCGCCTGGTACAGTCTGGGCCTGAGGTGAAGAAGCCTGGGTCCTCGGTGACGGTCTCCTGCCAGGCTTCTGGAGGCACCTTCAGCAGTTATGCTTTCACCTGGGTGCGCCAGGCCCCCGGACAAGGTCTTGAGTGGTTGGGCATGGTCACCCCAATCTTTGGTGAGGCCAAGTACTCACAAAGATTCGAGGGCAGAGTCACCATCACCGCGGACGAATCCACGAGCACAACCTCCATAGAATTGAGAGGCCTGACATCCGAAGACACGGCCATTTATTACTGTGCGCGAGATCGGCGCGCGGTTCCAATTGCCACGGACAACTGGTTAGACCCCTGGGGCCAGGGGACCCTGGTCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA

1456_P20 (PG20) gamma heavy chain variable region nucleotide sequence:

(SEQ ID NO: 101) CAGGTCCGCCTGGTACAGTCTGGGCCTGAGGTGAAGAAGCCTGGGTCCTCGGTGACGGTCTCCTGCCAGGCTTCTGGAGGCACCTTCAGCAGTTATGCTTTCACCTGGGTGCGCCAGGCCCCCGGACAAGGTCTTGAGTGGTTGGGCATGGTCACCCCAATCTTTGGTGAGGCCAAGTACTCACAAAGATTCGAGGGCAGAGTCACCATCACCGCGGACGAATCCACGAGCACAACCTCCATAGAATTGAGAGGCCTGACATCCGAAGACACGGCCATTTATTACTGTGCGCGAGATCGGCGCGCGGTTCCAATTGCCACGGACAACTGGTTAGACCCCTGGGGCCAGGGGACCCTGGTCACCGTCTCGAGC

1456_P20 (PG20) gamma heavy chain amino acid sequence: expressed proteinwith variable region in bold.

(SEQ ID NO: 16) QVRLVQSGPEVKKPGSSVTVSCQASGGTFSSYAFTWVRQAPGQGLEWLGMVTPIFGEAKYSQRFEGRVTITADESTSTTSIELRGLTSEDTAIYYCARDRRAVPIATDNWLDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK

1456_P20 (PG20) gamma heavy chain variable region amino acid sequence:(Kabat CDRs underlined, Chothia CDRs in bold italics)

(SEQ ID NO: 33) QVRLVQSGPEVKKPGSSVTVSCQA

WVRQAPGQGLEWL G

GRVTITADESTSTTSIELRGLTSEDTAIYYCARDR

WGQGTLVTVSS

1456_P20 (PG20) gamma heavy chain Kabat CDRs:

CDR 1: SGGTFSSYAFT (SEQ ID NO: 104) CDR 2: MVTPIFGEAKYSQRFE (SEQ ID NO:105) CDR 3: RAVPIATDNWLDP (SEQ ID NO: 102)

1456_P20 (PG20) gamma heavy chain Chothia CDRs:

CDR 1: SGGTFSSYAFT (SEQ ID NO: 104) CDR 2: MVTPIFGEAKYSQRFE (SEQ ID NO:105) CDR 3: RRAVPIATDNWLDP (SEQ ID NO: 103)

1456_P20 (PG20) kappa light chain nucleotide sequence: 1456_P20 κ1coding sequence (variable region in bold)

(SEQ ID NO: 17) ATGGACATGAGGGTCCCCGCTCAGCTCCTGGGGCTCCTGCTACTCTGGCTCCGAGGTGCCAGATGTGACATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTTGGCGACAGAGTCTCCATCACTTGCCGGGCGAGTCAGACCATTAACAACTACTTAAATTGGTATCAACAGACACCCGGGAAAGCCCCTAAACTCCTGATCTATGGTGCCTCCAATTTGCAAAATGGGGTCCCATCAAGGTTCAGCGGCAGTGGCTCTGGGACAGACTTCACTCTCACCATCAGCAGTCTGCAACCTGAGGATTTTGCAACTTACTACTGTCAACAGAGTTTCAGTACTCCGAGGACCTTCGGCCAAGGGACACGACTGGATATTAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGG GAGAGTGTTAG

1456_P20 (PG20) kappa light chain variable region nucleotide sequence:

(SEQ ID NO: 106) GACATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTTGGCGACAGAGTCTCCATCACTTGCCGGGCGAGTCAGACCATTAACAACTACTTAAATTGGTATCAACAGACACCCGGGAAAGCCCCTAAACTCCTGATCTATGGTGCCTCCAATTTGCAAAATGGGGTCCCATCAAGGTTCAGCGGCAGTGGCTCTGGGACAGACTTCACTCTCACCATCAGCAGTCTGCAACCTGAGGATTTTGCAACTTACTACTGTCAACAGAGTTTCAGTACTCCGAGGACCTTCGGCCAA GGGACACGACTGGATATTAAA

1456_P20 (PG20) kappa light chain amino acid sequence: expressed proteinwith variable region in bold.

(SEQ ID NO: 18) DIQLTQSPSSLSASVGDRVSITCRASQTINNYLNWYQQTPGKAPKLLIYGASNLQNGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSFSTPRTFGQGTRLDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC

1456_P20 (PG20) kappa light chain variable region amino acid sequence:(Kabat CDRs underlined, Chothia CDRs in bold italics)

(SEQ ID NO: 34) DIQLTQSPSSLSASVGDRVSITC

WYQQTPGKAPKLLIY

VPSRFSGSGS GTDFTLTISSLQPEDFATYYC

FGQGTRLDIK

1456_P20 (PG20) kappa light chain Kabat CDRs:

CDR 1: RASQTINNYLN (SEQ ID NO: 107) CDR 2: GASNLQNG (SEQ ID NO: 108) CDR3: QQSFSTPRT (SEQ ID NO: 42)

1456_P20 (PG20) kappa light chain Chothia CDRs:

CDR 1: RASQTINNYLN (SEQ ID NO: 107) CDR 2: GASNLQNG (SEQ ID NO: 108) CDR3: QQSFSTPRT (SEQ ID NO: 42)

1460_G14 (PGG14) gamma heavy chain nucleotide sequence: 1460_G14 γ1coding sequence (variable region in bold)

(SEQ ID NO: 19) ATGGACTGGATTTGGAGGTTCCTCTTGGTGGTGGCAGCAGCTACAGGTGTCCAGTCCCAGGTCCTGCTGGTGCAGTCTGGGACTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGTCAGGCTTCTGGAGGCGCCTTCAGTAGTTATGCTTTCAGCTGGGTGCGACAGGCCCCTGGACAGGGGCTTGAATGGATGGGCATGATCACCCCTGTCTTTGGTGAGACTAAATATGCACCGAGGTTCCAGGGCAGACTCACACTTACCGCGGAAGAATCCTTGAGCACCACCTACATGGAATTGAGAAGCCTGACATCTGATGACACGGCCTTTTATTATTGTACGAGAGATCGGCGCGTAGTTCCAATGGCCACAGACAACTGGTTAGACCCCTGGGGCCAGGGGACGCTGGTCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA

1460_G14 (PGG14) gamma heavy chain variable region nucleotide sequence:

(SEQ ID NO: 109) CAGGTCCTGCTGGTGCAGTCTGGGACTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGTCAGGCTTCTGGAGGCGCCTTCAGTAGTTATGCTTTCAGCTGGGTGCGACAGGCCCCTGGACAGGGGCTTGAATGGATGGGCATGATCACCCCTGTCTTTGGTGAGACTAAATATGCACCGAGGTTCCAGGGCAGACTCACACTTACCGCGGAAGAATCCTTGAGCACCACCTACATGGAATTGAGAAGCCTGACATCTGATGACACGGCCTTTTATTATTGTACGAGAGATCGGCGCGTAGTTCCAATGGCCACAGACAACTGGTTAGACCCCTGGGGCCAGGGGACGCTGGTCACCGTCTCGAGC

1460_G14 gamma heavy chain amino acid sequence: expressed protein withvariable region in bold.

(SEQ ID NO: 20) QVLLVQSGTEVKKPGSSVKVSCQASGGAFSSYAFSWVRQAPGQGLEWMGMITPVFGETKYAPRFQGRLTLTAEESLSTTYMELRSLTSDDTAFYYCTRDRRVVPMATDNWLDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPG

1460_G14 gamma heavy chain variable region amino acid sequence: (KabatCDRs underlined, Chothia CDRs in bold italics)

(SEQ ID NO: 35) QVLLVQSGTEVKKPGSSVKVSCQA

WVRQAPGQGLEWMG

GRLT LTAEESLSTTYMELRSLTSDDTAFYYCTRD

WGQGTLVTVSS

1460_G14 gamma heavy chain Kabat CDRs:

CDR 1: SGGAFSSYAFS (SEQ ID NO: 110) CDR 2: MITPVFGETKYAPRFQ (SEQ ID NO:111) CDR 3: RVVPMATDNWLDP (SEQ ID NO: 102)

1460_G14 gamma heavy chain Chothia CDRs:

CDR 1: SGGAFSSYAFS (SEQ ID NO: 110) CDR 2: MITPVFGETKYAPRFQ (SEQ ID NO:111) CDR 3: RRVVPMATDNWLDP (SEQ ID NO: 103)

1460_G14 (PGG14) kappa light chain nucleotide sequence: 1460_G14 κ1coding sequence (variable region in bold)

(SEQ ID NO: 21) ATGGACATGAGGGTCCCCGCTCAGCTCCTGGGGCTCCTGCTCCTCTGGCTCCGAGGTGCCACATGTGACATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGGGTCACCGTCACTTGCCGGGCGAGTCAGACCATACACACCTATTTAAATTGGTATCAGCAAATTCCAGGAAAAGCCCCTAAGCTCCTGATCTATGGTGCCTCCACCTTGCAAAGTGGGGTCCCGTCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAACAGTCTCCAACCTGAGGACTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCAAGGACCTTCGGCCAAGGGACACGACTGGATATTAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGG GAGAGTGTTAG

1460_G14 (PGG14) kappa light chain variable region nucleotide sequence:

(SEQ ID NO: 112) GACATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGGGTCACCGTCACTTGCCGGGCGAGTCAGACCATACACACCTATTTAAATTGGTATCAGCAAATTCCAGGAAAAGCCCCTAAGCTCCTGATCTATGGTGCCTCCACCTTGCAAAGTGGGGTCCCGTCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAACAGTCTCCAACCTGAGGACTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCAAGGACCTTCGGCCAA GGGACACGACTGGATATTAAA

1460_G14 kappa light chain amino acid sequence: expressed protein withvariable region in bold.

(SEQ ID NO: 22) DIQLTQSPSSLSASVGDRVTVTCRASQTIHTYLNWYQQIPGKAPKLLIYGASTLQSGVPSRFSGSGSGTDFTLTINSLQPEDFATYYCQQSYSTPRTFGQGTRLDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC

1460_G14 kappa light chain variable region amine acid sequence: (KabatCDRs underline, Chothia CDRs in bold italics)

(SEQ ID NO: 36) DIQLTQSPSSLSASVGDRVTVTC

WYQQIPGKAPKLLIY

VPSRFSGSG SGTDFTL TINSLQPEDFATYYC

FGQGTRLDIK

1460_G14 kappa light chain Kabat CDRs:

CDR 1: RASQTIHTYL (SEQ ID NO: 113) CDR 2: GASTLQSG (SEQ ID NO: 114) CDR3: QQSYSTPRT (SEQ ID NO: 43)

1460_G14 kappa light chain Chothia CDRs:

CDR 1: RASQTIHTYL (SEQ ID NO: 113) CDR 2: GASTLQSG (SEQ ID NO: 114) CDR3: QQSYSTPRT (SEQ ID NO: 43)

1495_C14 (PGC14) gamma heavy chain nucleotide sequence: 1495_C14 γ1coding sequence (variable region in bold)

(SEQ ID NO: 23) ATGGACTGGATTTGGAGGATCCTCCTCTTGGTGGCAGCAGCTACAGGCACCCTCGCCGACGGCCACCTGGTTCAGTCTGGGGTTGAGGTGAAGAAGACTGGGGCTACAGTCAAAATCTCCTGCAAGGTTTCTGGATACAGCTTCATCGACTACTACCTTCATTGGGTGCAACGGGCCCCTGGAAAAGGCCTTGAGTGGGTGGGACTTATTGATCCTGAAAATGGTGAGGCTCGATATGCAGAGAAGTTCCAGGGCAGAGTCACCATAATCGCGGACACGTCTATAGATACAGGCTACATGGAAATGAGGAGCCTGAAATCTGAGGACACGGCCGTGTATTTCTGTGCAGCAGGTGCCGTGGGGGCTGATTCCGGGAGCTGGTTCGACCCCTGGGGCCAGGGAACTCTGGTCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCT GTCTCCGGGTAAATGA

1495_C14 (PGC14) gamma heavy chain variable region nucleotide sequence:

(SEQ ID NO: 115) GACGGCCACCTGGTTCAGTCTGGGGTTGAGGTGAAGAAGACTGGGGCTACAGTCAAAATCTCCTGCAAGGTTTCTGGATACAGCTTCATCGACTACTACCTTCATTGGGTGCAACGGGCCCCTGGAAAAGGCCTTGAGTGGGTGGGACTTATTGATCCTGAAAATGGTGAGGCTCGATATGCAGAGAAGTTCCAGGGCAGAGTCACCATAATCGCGGACACGTCTATAGATACAGGCTACATGGAAATGAGGAGCCTGAAATCTGAGGACACGGCCGTGTATTTCTGTGCAGCAGGTGCCGTGGGGGCTGATTCCGGGAGCTGGTTCGACCCCTGGGGCCAGGGAACTCT GGTCACCGTCTCGAGC

1495_C14 (PGC14) gamma heavy chain amino acid sequence: expressedprotein with variable region in bold.

(SEQ ID NO: 24) DGHLVQSGVEVKKTGATVKISCKVSGYSFIDYYLHWVQRAPGKGLEWVGLIDPENGEARYAEKFQGRVTIIADTSIDTGYMEMRSLKSEDTAVYFCAAGAVGADSGSWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK

1495_C14 (PGC14) gamma heavy chain variable region amino acid sequence:(Kabat CDRs underlined, Chothia CDRs in bold italics)

(SEQ ID NO: 37) DGHLVQSGVEVKKTGATVKISCKV

WVQRAPGKGLEWVG

GRVTI IADTSIDTGYMEMRSLKSEDTAVYFCAAG

WGQGTLVTVSS

1495_C14 gamma heavy chain Kabat CDRs:

CDR 1: SGYSFIDYYLH (SEQ ID NO: 116) CDR 2: LIDPENGEARYAEKFQ (SEQ ID NO:117) CDR 3: AVGADSGSWFDP (SEQ ID NO: 118)

1495_C14 gamma heavy chain Chothia CDRs:

CDR 1: SGYSFIDYYLH (SEQ ID NO: 116) CDR 2: LIDPENGEARYAEKFQ (SEQ ID NO:117) CDR 3: AVGADSGSWFDP (SEQ ID NO: 118)

1495_C14 (PGC14) lambda light chain nucleotide sequence: 1495_C4 λ3coding sequence (variable region in bold)

(SEQ ID NO: 25) ATGGCCTGGATCCCTCTCTTCCTCGGCGTCCTTGCTTACTGCACAGATTCCGTAGTCTCCTATGAACTGACTCAGCCACCCTCAGTGTCCGTGTCCCCAGGACAGACAGCCAGCATCACCTGTTCTGGATCTAAATTGGGGGATAAATATGTTTCCTGGTATCAACTGAGGCCAGGCCAGTCCCCCATACTGGTCATGTATGAAAATGACAGGCGGCCCTCCGGGATCCCTGAGCGATTCTCCGGTTCCAATTCTGGCGACACTGCCACTCTGACCATCAGCGGGACCCAGGCTTTGGATGAGGCTGACTTCTACTGTCAGGCGTGGGAGACCACCACCACCACTTTTGTTTTCTTCGGCGGAGGGACCCAGCTGACCGTTCTAGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTACCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAAAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTT CATAG

1495_C14 (PGC14) lambda light chain variable region nucleotide sequence:

(SEQ ID NO: 119) TCCTATGAACTGACTCAGCCACCCTCAGTGTCCGTGTCCCCAGGACAGACAGCCAGCATCACCTGTTCTGGATCTAAATTGGGGGATAAATATGTTTCCTGGTATCAACTGAGGCCAGGCCAGTCCCCCATACTGGTCATGTATGAAAATGACAGGCGGCCCTCCGGGATCCCTGAGCGATTCTCCGGTTCCAATTCTGGCGACACTGCCACTCTGACCATCAGCGGGACCCAGGCTTTGGATGAGGCTGACTTCTACTGTCAGGCGTGGGAGACCACCACCACCACTTTTGTTTTCTTCGGCGGAGGGACCCAGCTGACCGTTCTA

1495_C14 (PGC14) lambda light chain amino acid sequence: expressedprotein with variable region in bold.

(SEQ ID NO: 26) SYELTQPPSVSVSPGQTASITCSGSKLGDKYVSWYQLRPGQSPILVMYENDRRPSGIPERFSGSNSGDTATLTISGTQALDEADFYCQAWETTTTTFVFFGGGTQLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTH EGSTVEKTVAPTECS

1495_C14 (PGC14) lambda light chain variable region amino acid sequence:(Kabat CDRs underlined Chothia CDRs in bold italics)

(SEQ ID NO: 38) SYELTQPPSVSVSPGQTASITC

WYQLRPGQSPILVMY

IPERFSGSNSGDTAT LTISGTQALDEADFYC

FGGGTQLTVL

1495_C14 (PGC14) lambda light chain Kabat CDRs:

CDR 1: SGSKLGDKYVS (SEQ ID NO: 120) CDR 2: ENDRRPSG (SEQ ID NO: 121) CDR3: QAWETTTTTFVF (SEQ ID NO: 44)

1495_C14 (PGC14) lambda light chain Chothia CDRs:

CDR 1: SGSKLGDKYVS (SEQ ID NO: 120) CDR 2: ENDRRPSG (SEQ ID NO: 121) CDR3: QAWETTTTTFVF (SEQ ID NO: 44)

1496_C09 (PG9) gamma heavy chain nucleotide sequence: 1496_C09 γ3 codingsequence (variable region in bold)

(SEQ ID NO: 27) ATGGAGTTTGGGCTGAGCTGGGTTTTCCTCGTTGCTTTCTTAAGAGGTGTCCAGTGTCAGCGATTAGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGTCGTCCCTGAGACTCTCCTGTGCAGCGTCCGGATTCGACTTCAGTAGACAAGGCATGCACTGGGTCCGCCAGGCTCCAGGCCAGGGGCTGGAGTGGGTGGCATTTATTAAATATGATGGAAGTGAGAAATATCATGCTGACTCCGTATGGGGCCGACTCAGCATCTCCAGAGACAATTCCAAGGATACGCTTTATCTCCAAATGAATAGCCTGAGAGTCGAGGACACGGCTACATATTTTTGTGTGAGAGAGGCTGGTGGGCCCGACTACCGTAATGGGTACAACTATTACGATTTCTATGATGGTTATTATAACTACCACTATATGGACGTCTGGGGCAAAGGGACCACGGTCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGG GTAAATGA

1496_C09 (PG9) gamma heavy chain variable region nucleotide sequence:

(SEQ ID NO: 122) CAGCGATTAGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGTCGTCCCTGAGACTCTCCTGTGCAGCGTCCGGATTCGACTTCAGTAGACAAGGCATGCACTGGGTCCGCCAGGCTCCAGGCCAGGGGCTGGAGTGGGTGGCATTTATTAAATATGATGGAAGTGAGAAATATCATGCTGACTCCGTATGGGGCCGACTCAGCATCTCCAGAGACAATTCCAAGGATACGCTTTATCTCCAAATGAATAGCCTGAGAGTCGAGGACACGGCTACATATTTTTGTGTGAGAGAGGCTGGTGGGCCCGACTACCGTAATGGGTACAACTATTACGATTTCTATGATGGTTATTATAACTACCACTATATGGACGTCTGGGGCAAAGGGACCACGGTCACCG TCTCGAGC

1496_C09 (PG9) gamma heavy chain amino acid sequence: expressed proteinwith variable region in bold.

(SEQ ID NO: 28) QRLVESGGGVVQPGSSLRLSCAASGFDFSRQGMHWVRQAPGQGLEWVAFIKYDGSEKYHADSVWGRLSISRDNSKDTLYLQMNSLRVEDTATYFCVREAGGPDYRNGYNYYDFYDGYYNYHYMDVWGKGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK

1496_C09 (PG9) gamma heavy chain variable region amino acid sequence:(Kabat CDRs underlined, Chothia CDRs in bold italics)

(SEQ ID NO: 39) QRLVESGGGVVQPGSSLRLSCAA

WVRQAPGQGLEWVA

GRLSI SRDNSKDTLYLQMNSLRVEDTATYFCVR

WGKGTTVTVSS

1496_C09 (PG9) gamma heavy chain Kabat CDRs:

CDR 1: (SEQ ID NO: 123) SGFDFSRQGMH CDR 2: (SEQ ID NO: 124)FIKYDGSEKYHADSVW CDR 3: (SEQ ID NO: 7) EAGGPDYRNGYNYYDFYDGYYNYHYMDV

1496_C09 (PG9) gamma heavy chain Chothia CDRs:

CDR 1: (SEQ ID NO: 123) SGFDFSRQGMH CDR 2: (SEQ ID NO: 124)FIKYDGSEKYHADSVW CDR 3: (SEQ ID NO: 7) EAGGPDYRNGYNYYDFYDGYYNYHYMDV

1496_C09 (PG9) lambda light chain nucleotide sequence: 1496_C09 λ2coding sequence (variable region in bold)

(SEQ ID NO: 29) ATGGCCTGGGCTCTGCTTTTCCTCACCCTCCTCACTCAGGGCACAGGGTCCTGGGCCCAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTGCAATGGAACCAGCAATGATGTTGGTGGCTATGAATCTGTCTCCTGGTACCAACAACATCCCGGCAAAGCCCCCAAAGTCGTGATTTATGATGTCAGTAAACGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCCGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGACGAGGGTGACTATTACTGCAAGTCTCTGACAAGCACGAGACGTCGGGTTTTCGGCACTGGGACCAAGCTGACCGTTCTAGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTACCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAAAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAAT GTTCATAG

1496_C09 (PG9) lambda light chain variable region nucleotide sequence:

(SEQ ID NO: 125) CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTGCAATGGAACCAGCAATGATGTTGGTGGCTATGAATCTGTCTCCTGGTACCAACAACATCCCGGCAAAGCCCCCAAAGTCGTGATTTATGATGTCAGTAAACGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCCGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGACGAGGGTGACTATTACTGCAAGTCTCTGACAAGCACGAGACGTCGGGTTTTCGGCACTGGGACCAAGCTGACCGTTCTA

1496_C09 (PG9) lambda light chain amino acid sequence: expressed proteinwith variable region in bold.

(SEQ ID NO: 30) QSALTQPASVSGSPGQSITISCNGTSNDVGGYESVSWYQQHPGKAPKVVIYDVSKRPSGVSNRFSGSKSGNTASLTISGLQAEDEGDYYCKSLTSTRRRVFGTGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVT HEGSTVEKTVAPTECS

1496_C09 (PG9) lambda light chain variable region amino acid sequence:(Kabat CDRs underlined, Chothia CDRs in bold italics)

(SEQ ID NO: 40) QSALTQPASVSGSPGQSITISC

WYQQHPGKAPKVVIY

VSNRFSGSKS GNTASLTISGLQAEDEGDYYC

FGTGTKLTVL

1496_C09 (PG9) lambda light chain Kabat CDRs:

CDR 1: NGTSNDVGGYESVS (SEQ ID NO: 126) CDR 2: DVSKRPSG (SEQ ID NO: 127)CDR 3: KSLTSTRRRV (SEQ ID NO: 45)

1496_C09 (PG9) lambda light chain Chothia CDRs:

CDR 1: NGTSNDVGGYESVS (SEQ ID NO: 126) CDR 2: DVSKRPSG (SEQ ID NO: 127)CDR 3: KSLTSTRRRV (SEQ ID NO: 45)

The PG16 antibody includes a heavy chain variable region (SEQ ID NO:31), encoded by the nucleic acid sequence shown in SEQ ID NO: 99, and alight chain variable region (SEQ ID NO: 32) encoded by the nucleic acidsequence shown in SEQ ID NO: 100.

The heavy chain CDRs of the PG16 antibody have the following sequencesper Kabat and Chothia definitions: SGFTFHKYGMH (SEQ ID NO: 88),LISDDGMRKYHSDSMW (SEQ ID NO: 89), and EAGGPIWHDDVKYYDFNDGYYNYHYMDV (SEQID NO: 6). The light chain CDRs of the PG16 antibody have the followingsequences per Kabat and Chothia definitions: NGTSSDVGGFDSVS (SEQ ID NO:97), DVSHRPSG (SEQ ID NO: 95), and SSLTDRSHRI (SEQ ID NO: 41).

The PG20 antibody includes a heavy chain variable region (SEQ ID NO:33), encoded by the nucleic acid sequence shown in SEQ ID NO: 101, and alight chain variable region (SEQ ID NO: 34) encoded by the nucleic acidsequence shown in SEQ ID NO: 106.

The heavy chain CDRs of the PG20 antibody have the following sequencesper Kabat definition: SGGTFSSYAFT (SEQ ID NO: 104), MVTPIFGEAKYSQRFE(SEQ ID NO: 105), and RAVPIATDNWLDP (SEQ ID NO: 102). The light chainCDRs of the PG20 antibody have the following sequences per Kabatdefinition: RASQTINNYLN (SEQ ID NO: 107), GASNLQNG (SEQ ID NO: 108), andQQSFSTPRT (SEQ ID NO: 42).

The heavy chain CDRs of the PG20 antibody have the following sequencesper Chothia definition: SGGTFSSYAFT (SEQ ID NO: 104), MVTPIFGEAKYSQRFE(SEQ ID NO: 105), and RRAVPIATDNWLDP (SEQ ID NO: 103). The light chainCDRs of the PG20 antibody have the following sequences per Chothiadefinition: RASQTINNYLN (SEQ ID NO: 107), GASNLQNG (SEQ ID NO: 108), andQQSFSTPRT (SEQ ID NO: 42).

The PGG14 antibody includes a heavy chain variable region (SEQ ID NO:35), encoded by the nucleic acid sequence shown in SEQ ID NO: 109, and alight chain variable region (SEQ ID NO: 36) encoded by the nucleic acidsequence shown in SEQ ID NO: 112.

The heavy chain CDRs of the PGG14 antibody have the following sequencesper Kabat definition: SGGAFSSYAFS (SEQ ID NO: 110), MITPVFGETKYAPRFQ(SEQ ID NO: 111), and RVVPMATDNWLDP (SEQ ID NO: 102). The light chainCDRs of the PGG14 antibody have the following sequences per Kabatdefinition: RASQTIHTYL (SEQ ID NO: 113), GASTLQSG (SEQ ID NO: 114), andQQSYSTPRT (SEQ ID NO: 43).

The heavy chain CDRs of the PGG14 antibody have the following sequencesper Chothia definition: SGGAFSSYAFS (SEQ ID NO: 110), MITPVFGETKYAPRFQ(SEQ ID NO: 111), RRVVPMATDNWLDP (SEQ ID NO: 103). The light chain CDRsof the PGG14 antibody have the following sequences per Chothiadefinition: RASQTIHTYL (SEQ ID NO: 113), GASTLQSG (SEQ ID NO: 114), andQQSYSTPRT (SEQ ID NO: 43).

The PGC14 antibody includes a heavy chain variable region (SEQ ID NO:37), encoded by the nucleic acid sequence shown in SEQ ID NO: 115, and alight chain variable region (SEQ ID NO: 38) encoded by the nucleic acidsequence shown in SEQ ID NO: 119.

The heavy chain CDRs of the PGC14 antibody have the following sequencesper Kabat and Chothia definitions: SGYSFIDYYLH (SEQ ID NO: 116),LIDPENGEARYAEKFQ (SEQ ID NO: 117), and AVGADSGSWFDP (SEQ ID NO: 118).The light chain CDRs of the PGC14 antibody have the following sequencesper Kabat and Chothia definitions: SGSKLGDKYVS (SEQ ID NO: 120),ENDRRPSG (SEQ ID NO: 121), and QAWETTTTTFVF (SEQ ID NO: 44).

The PG9 antibody includes a heavy chain variable region (SEQ ID NO: 39),encoded by the nucleic acid sequence shown in SEQ ID NO: 122, and alight chain variable region (SEQ ID NO: 40) encoded by the nucleic acidsequence shown in SEQ ID NO: 125.

The heavy chain CDRs of the PG9 antibody have the following sequencesper Kabat and Chothia definitions: SGFDFSRQGMH (SEQ ID NO: 123),FIKYDGSEKYHADSVW (SEQ ID NO: 124), and EAGGPDYRNGYNYYDFYDGYYNYHYMDV (SEQID NO: 7). The light chain CDRs of the PG9 antibody have the followingsequences per Kabat and Chothia definitions: NGTSNDVGGYESVS (SEQ ID NO:126), DVSKRPSG (SEQ ID NO: 127), and KSLTSTRRRV (SEQ ID NO: 45).

TABLE 6A Heavy Chain Variable Region Protein Alignment 10 20 301495_C14_G1 ref D G H L V Q S G V E V K K T G A T V K I S C K V S G Y SF I D Y Y L H 1460_G14_G1 ref Q V L L V Q S G T E V K K P G S S V K V SC Q A S G G A F S S Y A F S 1456_P20_G1 ref Q V R L V Q S G P E V K K PG S S V T V S C Q A S G G T F S S Y A F T 1443_C16_G3 ref Q E Q L V E SG G G V V Q P G G S L R L S C L A S G F T F H K Y G M H 1496_C09_G3 refQ — R L V E S G G G V V Q P G S S L R L S C A A S G F D F S R Q G M H 4050 60 70 1495_C14_G1 ref W V Q R A P G K G L E W V G L I D P E N G E A RY A E K F Q G R V T I 1460_G14_G1 ref W V R Q A P G Q G L E W M G M I TP V F G E T K Y A P R F Q G R L T L 1456_P20_G1 ref W V R Q A P G Q G LE W L G M V T P I F G E A K Y S Q R F E G R V T I 1443_C16_G3 ref W V RQ A P G K G L E W V A L I S D D G M R K Y H S D S M W G R V T I1496_C09_G3 ref W V R Q A P G Q G L E W V A F I K Y D G S E K Y H A D SV W G R L S I 80 90 100 1495_C14_G1 ref I A D T S I D T G Y M E M R S LK S E D T A V Y F C A A G — — — — — — — 1460_G14_G1 ref T A E E S L S TT Y M E L R S L T S D D T A F Y Y C T R D R R — — — — — 1456_P20_G1 refT A D E S T S T T S I E L R G L T S E D T A I Y Y C A R D R R — — — — —1443_C16_G3 ref S R D N S K N T L Y L Q F S S L K V E D T A M F F C A RE A G G P I W H 1496_C09_G3 ref S R D N S K D T L Y L Q M N S L R V E DT A T Y F C V R E A G G P D Y R 110 120 130 1495_C14_G1 ref — — — — — —— — A V G A D S G S W F D P W G Q G T L V T V S S 1460_G14_G1 ref — — —— — — — — V V P M A T D N W L D P W G Q G T L V T V S S 1456_P20_G1 ref— — — — — — — — A V P I A T D N W L D P W G Q G T L V T V S S1443_C16_G3 ref D D V K Y Y D F N D G Y Y N Y H Y M D V W G K G T T V TV S S 1496_C09_G3 ref N G Y N Y Y D F Y D G Y Y N Y H Y M D V W G K G TT V T V S S

TABLE 6B Light Chain Variable Region Protein Alignment 10 20 301495_C14_L3 ref S Y E L T Q — P P S V S V S P G Q T A S I T C S G S K —— — L G D K Y V S W 1460_G14_K1 ref D I Q L T Q S P S S L S A S V G D RV T V T C R A S Q T — — — I H T Y L N W 1456_P20_K1 ref D I Q L T Q S PS S L S A S V G D R V S I T C R A S Q T — — — I N N Y L N W 1443_C16_L2ref Q S A L T Q — P A S V S G S P G Q T I T I S C N G T S S D V G G F DS V S W 1496_C09_L2 ref Q S A L T Q — P A S V S G S P G Q S I T I S C NG T S N D V G G Y E S V S W 40 50 60 70 1495_C14_L3 ref Y Q L R P G Q SP I L V M Y E N D R R P S G I P E R F S G S N S G D T A T L 1460_G14_K1ref Y Q Q I P G K A P K L L I Y G A S T L Q S G V P S R F S G S G S G TD F T L 1456_P20_K1 ref Y Q Q T P G K A P K L L I Y G A S N L Q N G V PS R F S G S G S G T D F T L 1443_C16_L2 ref Y Q Q S P G K A P K V M V FD V S H R P S G I S N R F S G S K S G N T A S L 1496_C09_L2 ref Y Q Q HP G K A P K V V I Y D V S K R P S G V S N R F S G S K S G N T A S L 8090 100 110 1495_C14_L3 ref T I S G T Q A L D E A D F Y C Q A W E T T T TT F V F F G G G T Q L T V L G 1460_G14_K1 ref T I N S L Q P E D F A T YY C Q Q — — — S Y S T P R T F G Q G T R L D I K — 1456_P20_K1 ref T I SS L Q P E D F A T Y Y C Q Q — — — S F S T P R T F G Q G T R L D I K —1443_C16_L2 ref T I S G L H I E D E G D Y F C S S — — L T D R S H R I FG G G T K V T V L G 1496_C09_L2 ref T I S G L Q A E D E G D Y Y C K S —— L T S T R R R V F G T G T K L T V L G

The sequences of sister clones to human monoclonal antibody 1443_C16(PG16) were determined, including the sequences of the variable regionsof the Gamma heavy and Kappa or Lambda light chains. In addition, thesequence of each of the polynucleotides encoding the antibody sequenceswas determined. Shown below are the polypeptide and polynucleotidesequences of the gamma heavy chains and kappa light chains, with thesignal peptides at the N-terminus (or 5′ end) and the constant regionsat the C-terminus (or 3′ end) of the variable regions, which are shownin bolded text.

1469_M23 gamma heavy chain nucleotide sequence: 1469_M23 γ3 codingsequence (variable region in bold)

(SEQ ID NO: 138) ATGGAGTTTGGGCTGAGCTGGGTTTTCCTCGCAACTCTGTTAAGAGTTGTGAAGTGTCAGGAAAAACTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCGGGGGGGTCCCTGAGACTCTCCTGTTTAGCGTCTGGATTCACCTTTCACAAATATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGCCTGGAGTGGGTGGCACTCATCTCAGATGACGGAATGAGGAAATATCATTCAGACTCCATGTGGGGCCGAGTCACCATCTCCAGAGACAATTCCAAGAACACTCTATATCTGCAATTCaGCAGCCTGAAAGTCGAAGACACGGCTATGTTCTTCTGTGCGAGAGAGGCTGGTGGGCCAATCTGGCATGACGACGTCAAATATTACGATTTTAATGACGGCTACTACAACTACCACTACATGGACGTCTGGGGCAAGGGGACCACGGTCACCGtCTCCTCAGCGTCGACCAAGGGCCCATCGGTCTTCCCTCTGGCACCATCATCCAAGTCGACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTC CGGGTAAATGA

1469_M23 gamma heavy chain variable region nucleotide sequence:

(SEQ ID NO: 128) CAGGAAAAACTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCGGGGGGGTCCCTGAGACTCTCCTGTTTAGCGTCTGGATTCACCTTTCACAAATATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGCCTGGAGTGGGTGGCACTCATCTCAGATGACGGAATGAGGAAATATCATTCAGACTCCATGTGGGGCCGAGTCACCATCTCCAGAGACAATTCCAAGAACACTCTATATCTGCAATTCaGCAGCCTGAAAGTCGAAGACACGGCTATGTTCTTCTGTGCGAGAGAGGCTGGTGGGCCAATCTGGCATGACGACGTCAAATATTACGATTTTAATGACGGCTACTACAACTACCACTACATGGACGTCTGGGGCAAGGGGACCACGGTCA CCGtCTCCTCA

1469_M23 gamma heavy chain amino acid sequence: expressed protein withvariable region in bold.

(SEQ ID NO: 139) QEKLVESGGGVVQPGGSLRLSCLASGFTFHKYGMHWVRQAPGKGLEWVALISDDGMRKYHSDSMWGRVTISRDNSKNTLYLQFSSLKVEDTAMFFCAREAGGPIWHDDVKYYDFNDGYYNYHYMDVWGKGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK

1469_M23 gamma heavy chain variable region amino acid sequence: (KabatCDRs underlined, Chothia CDRs in bold italics)

(SEQ ID NO: 140) QEKLVESGGGVVQPGGSLRLSCLA

WVRQAPGKGLEWVA

GRVTISRDNSKNTLYLQFSSLKVEDTAMFFCAR

WGKGTTVTVSS

1469_M23 gamma heavy chain Kabat CDRs:

CDR 1: (SEQ ID NO: 88) SGFTFHKYGMH CDR 2: (SEQ ID NO: 89)LISDDGMRKYHSDSMW CDR 3: (SEQ ID NO: 6) EAGGPIWHDDVKYYDFNDGYYNYHYMDV

1469_M23 gamma heavy chain Chothia CDRs:

CDR 1: (SEQ ID NO: 88) SGFTFHKYGMH CDR 2: (SEQ ID NO: 89)LISDDGMRKYHSDSMW CDR 3: (SEQ ID NO: 6) EAGGPIWHDDVKYYDFNDGYYNYHYMDV

1469_M23 lambda light chain nucleotide sequence: 1469_M23 λ2 codingsequence (variable region in bold)

(SEQ ID NO: 141) ATGGCCTGGGCTCTGCTATTCCTCACCCTCTTCACTCAGGGCACAGGGTCCTGGGGCCAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGACGATCACCATCTCCTGCAATGGAACCAGAAGTGACGTTGGTGGATTTGACTCTGTCTCCTGGTACCAACAATCCCCAGGGAGAGCCCCCAAAGTCATGGTTTTTGATGTCAGTCATCGGCCCTCAGGTATCTCTAATCGCTTCTCTGGCTCCAAGTCCGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCACATTGAGGACGAGGGCGATTATTTCTGCTCTTCACTGACAGACAGAAGCCATCGCATATTCGGCGGCGGGACCAAGCTGACCGTTCTAGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTACCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAAAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAAT GTTCATAG

1469_M23 lambda light chain variable region nucleotide sequence:

(SEQ ID NO: 129) CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGACGATCACCATCTCCTGCAATGGAACCAGAAGTGACGTTGGTGGATTTGACTCTGTCTCCTGGTACCAACAATCCCCAGGGAGAGCCCCCAAAGTCATGGTTTTTGATGTCAGTCATCGGCCCTCAGGTATCTCTAATCGCTTCTCTGGCTCCAAGTCCGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCACATTGAGGACGAGGGCGATTATTTCTGCTCTTCACTGACAGACAGAAGCCATCGCATATTCGGCGGCGGGACCAAGCTGACCGTTCTA

1469_M23 lambda light chain amino acid sequence: expressed protein withvariable region in bold.

(SEQ ID NO: 142) QSALTQPASVSGSPGQTITISCNGTRSDVGGFDSVSWYQQSPGRAPKVMVFDVSHRPSGISNRFSGSKSGNTASLTISGLHIEDEGDYFCSSLTDRSHRIFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVT HEGSTVEKTVAPTECS

1469_M23 lambda light chain variable region amino acid sequence: (KabatCDRs underlined, Chothia CDRs in bold italics)

(SEQ ID NO: 96) QSALTQPASVSGSPGQTITISC

WYQQSPGRAPKVMV F

ISNRFSGSKSGNTASLTISGLHIEDEGDYFC

FGGGT KLTVL

1469_M23 lambda light chain Kabat CDRs:

CDR 1: NGTRSDVGGFDSVS (SEQ ID NO: 92) CDR 2: DVSHRPSG (SEQ ID NO: 95)CDR 3: SSLTDRSHRI (SEQ ID NO: 41)

1469_M23 lambda light chain Chothia CDRs:

CDR 1: NGTRSDVGGFDSVS (SEQ ID NO: 92) CDR 2: DVSHRPSG (SEQ ID NO: 95)CDR 3: SSLTDRSHRI (SEQ ID NO: 41)

1456_A12 gamma heavy chain nucleotide sequence: 1456 A12 γ3 codingsequence (variable region in bold)

(SEQ ID NO: 46) ATGGAGTTTGGGCTGAGCTGGGTTTTCCTCGCAACTCTGTTAAGAGTTGTGAAGTGTCACGAACAACTGGTGGAGGCCGGGGGAGGCGTGGTCCAGCCGGGGGGGTCCCTGAGACTCTCCTGTTTAGCGTCTGGATTCACGTTTCACAAATATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGCCTGGAGTGGGTGGCACTCATCTCAGATGACGGAATGAGGAAATATCATTCAGACTCCATGTGGGGCCGAGTCACCATCTCCAGAGACAATTCCAAGAACACTCTTTATCTGCAATTCAGCAGCCTGAGAGTCGAAGACACGGCTATGTTCTTCTGTGCGAGAGAGGCCGGTGGGCCAATCTGGCATGACGACGTCAAATATTACGATTTTAATGACGGCTACTACAACTATCACTACATGGACGTCTGGGGCAAGGGGACCAAGGTCACCGTCTCCTCAGCGTCGACCAAGGGCCCATCGGTCTTCCCTCTGGCACCATCATCCAAGTCGACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTC CGGGTAAATGA

1456_A12 gamma heavy chain variable region nucleotide sequence:

(SEQ ID NO: 130) CACGAACAACTGGTGGAGGCCGGGGGAGGCGTGGTCCAGCCGGGGGGGTCCCTGAGACTCTCCTGTTTAGCGTCTGGATTCACGTTTCACAAATATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGCCTGGAGTGGGTGGCACTCATCTCAGATGACGGAATGAGGAAATATCATTCAGACTCCATGTGGGGCCGAGTCACCATCTCCAGAGACAATTCCAAGAACACTCTTTATCTGCAATTCAGCAGCCTGAGAGTCGAAGACACGGCTATGTTCTTCTGTGCGAGAGAGGCCGGTGGGCCAATCTGGCATGACGACGTCAAATATTACGATTTTAATGACGGCTACTACAACTATCACTACATGGACGTCTGGGGCAAGGGGACCAAGGTCA CCGTCTCCTCA

1456_A12 gamma heavy chain amino acid sequence: expressed protein withvariable region in bold.

(SEQ ID NO: 47) HEQLVEAGGGVVQPGGSLRLSCLASGFTFHKYGMHWVRQAPGKGLEWVALISDDGMRKYHSDSMWGRVTISRDNSKNTLYLQFSSLRVEDTAMFFCAREAGGPIWHDDVKYYDFNDGYYNYHYMDVWGKGTKVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK

1456_A12 gamma heavy chain variable region amino acid sequence: (KabatCDRs underlined, Chothia CDRs in bold italics)

(SEQ ID NO: 48) HEQLVEAGGGVVQPGGSLRLSCLA

WVRQAPGKGLEWVA

GRVTISRDNSKNTLYLQFSSLRVEDTAM FFCAR

VWGKGTKVTVSS

1456_A12 gamma heavy chain Kabat CDRs:

CDR 1: (SEQ ID NO: 88) SGFTFHKYGMH CDR 2: (SEQ ID NO: 89)LISDDGMRKYHSDSMW CDR 3: (SEQ ID NO: 6) EAGGPIWHDDVKYYDFNDGYYNYHYMDV

1456_A12 gamma heavy chain Chothia CDRs:

CDR 1: (SEQ ID NO: 88) SGFTFHKYGMH CDR 2: (SEQ ID NO: 89)LISDDGMRKYHSDSMW CDR 3: (SEQ ID NO: 6) EAGGPIWHDDVKYYDFNDGYYNYHYMDV

1456_A12 lambda light chain nucleotide sequence: 1456_A12 λ2 codingsequence (variable region in bold)

(SEQ ID NO: 49) ATGGCCTGGGCTTGCTATTCCTCACCCTCTTCACTCAGGGCACAGGGTCCTGGGGCCAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGACGATCACCATCTCCTGCAATGGAACCAGCCGTGACGTTGGTGGATTTGACTCTGTCTCCTGGTATCAACAATCCCCAGGGAAAGCCCCCAAAGTCATGGTTTTTGATGTCAGTCATCGGCCCTCAGGTATGTCTAATCGCTTCTCTGGCTCCAAGTCCGGCAACACGGCCTCCCTGACCATTTCTGGGCTCCACATTGAGGACGAGGGCGATTATTTCTGCTCTTCATTGACAGACAGAAGCCATCGCATATTCGGCGGCGGGACCAAGCTGACCGTTCTAGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTACCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAAAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATG TTCATAG

1456_A12 lambda light chain variable region nucleotide sequence:

(SEQ ID NO: 131) CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGACGATCACCATCTCCTGCAATGGAACCAGCCGTGACGTTGGTGGATTTGACTCTGTCTCCTGGTATCAACAATCCCCAGGGAAAGCCCCCAAAGTCATGGTTTTTGATGTCAGTCATCGGCCCTCAGGTATGTCTAATCGCTTCTCTGGCTCCAAGTCCGGCAACACGGCCTCCCTGACCATTTCTGGGCTCCACATTGAGGACGAGGGCGATTATTTCTGCTCTTCATTGACAGACAGAAGCCATCGCATATTCGGCGGCGGGACCAAGCTGACCGTTCTA

1456_A12 lambda light chain amino acid sequence: expressed protein withvariable region in bold.

(SEQ ID NO: 50) QSALTQPASVSGSPGQTITISCNGTSRDVGGFDSVSWYQQSPGKAPKVMVFDVSHRPSGMSNRFSGSKSGNTASLTISGLHIEDEGDYFCSSLTDRSHRIFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVT HEGSTVEKTVAPTECS

1456_A12 lambda light chain variable region amino acid sequence: (KabatCDRs underline, Chothia CDRs in bold italics)

(SEQ ID NO: 51) QSALTQPASVSGSPGQTITISC

WYQQSPGKAPKVMVF

MSNRFS GSKSGNTASLTISGLHIEDEGDYFC

FGGGTKLTVL

1456_A12 lambda light chain Kabat CDRs:

CDR 1: NGTSRDVGGFDSVS (SEQ ID NO: 93) CDR 2: DVSHRPSG (SEQ ID NO: 95)CDR 3: SSLTDRSHRI (SEQ ID NO: 41)

1456_A12 lambda light chain Chothia CDRs:

CDR 1: NGTSRDVGGFDSVS (SEQ ID NO: 93) CDR 2: DVSHRPSG (SEQ ID NO: 95)CDR 3: SSLTDRSHRI (SEQ ID NO: 41)

1503_H05 gamma heavy chain nucleotide sequence: 1503_H05 γ3 codingsequence (variable region in bold)

(SEQ ID NO: 52) ATGGAGTTTGGCTGAGCTGGGTTTTCCTCGCAACTCTGTTAAGAGTTGTGAAGTGTCAGGAAAAACTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCGGGGGGGTCCCTGAGACTCTCCTGTTTAGCGTCTGGATTCACCTTTCACAAATATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGCCTGGAGTGGGTGGCACTCATCTCAGATGACGGAATGAGGAAATATCATTCAGACTCCATGTGGGGCCGAGTCACCATCTCCAGAGACAATTCCAAGAACACTTTATATCTGCAATTCAGCAGCCTGAAAGTCGAAGACACGGCTATGTTCTTCTGTGCGAGAGAGGCTGGTGGGCCAATCTGGCATGACGACGTCAAATATTACGATTTTAATGACGGCTACTACAATTACCACTACATGGACGTCTGGGGCAAGGGGACCATTGTCACCGTCTCCTCAGCGTCGACCAAGGGCCCATCGGTCTTCCCTCTGGCACCATCATCCAAGTCGACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCC GGGTAAATGA

1503_H05 gamma heavy chain variable region nucleotide sequence:

(SEQ ID NO: 132) CAGGAAAAACTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCGGGGGGGTCCCTGAGACTCTCCTGTTTAGCGTCTGGATTCACCTTTCACAAATATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGCCTGGAGTGGGTGGCACTCATCTCAGATGACGGAATGAGGAAATATCATTCAGACTCCATGTGGGGCCGAGTCACCATCTCCAGAGACAATTCCAAGAACACTTTATATCTGCAATTCAGCAGCCTGAAAGTCGAAGACACGGCTATGTTCTTCTGTGCGAGAGAGGCTGGTGGGCCAATCTGGCATGACGACGTCAAATATTACGATTTTAATGACGGCTACTACAATTACCACTACATGGACGTCTGGGGCAAGGGGACCATTGTCA CCGTCTCCTCA

1503_H05 gamma heavy chain amino acid sequence: expressed protein withvariable region in bold.

(SEQ ID NO: 53) QEKLVESGGGVVQPGGSLRLSCLASGFTFHKYGMHWVRQAPGKGLEWVALISDDGMRKYHSDSMWGRVTISRDNSKNTLYLQFSSLKVEDTAMFFCAREAGGPIWHDDVKYYDFNDGYYNYHYMDVWGKGTIVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK.

1503_H05 gamma heavy chain variable region amino acid sequence: (KabatCDRs underlined, Chothia CDRs in bold italics)

(SEQ ID NO: 54) QEKLVESGGGVVQPGGSLRLSCLA

WVRQAPGKGLEWVA

GRVTISRDNSKNTLYLQFSSLKVEDTAMFFCAR

WGKGTIVTVSS

1503_H05 gamma heavy chain Kabat CDRs:

CDR 1: (SEQ ID NO: 88) SGFTFHKYGMH CDR 2: (SEQ ID NO: 89)LISDDGMRKYHSDSMW CDR 3: (SEQ ID NO: 6) EAGGPIWHDDVKYYDFNDGYYNYHYMDV

1503_H05 gamma heavy chain Chothia CDRs:

CDR 1: (SEQ ID NO: 88) SGFTFHKYGMH CDR 2: (SEQ ID NO: 89)LISDDGMRKYHSDSMW CDR 3: (SEQ ID NO: 6) EAGGPIWHDDVKYYDFNDGYYNYHYMDV

1503_H05 lambda light chain nucleotide sequence: 1503_H05 λ2 codingsequence (variable region in bold)

(SEQ ID NO: 55) ATGGCCTGGGCTTGCTATTCCTCACCCTCTTCACTCAGGGCACAGGGTCCTGGGGCCAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGACGATCACCATCTCCTGCAATGGAACCAGAAGTGACGTTGGTGGATTTGACTCTGTCTCCTGGTACCAACAATCCCCAGGGAAAGCCCCCAAAGTCATGGTTTTTGATGTCAGTCATCGGCCCTCAGGTATCTCTAATCGCTTCTCTGGCTCCAAGTCCGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCACATTGAGGACGAGGGCGATTATTTCTGCTCTTCACTGACAGACAGAAGCCATCGCATATTCGGCGGCGGGACCAAGGTGACCGTTCTAGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTACCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAAAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATG TTCATAG

1503_H05 lambda light chain variable region nucleotide sequence:

(SEQ ID NO: 133) CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGACGATCACCATCTCCTGCAATGGAACCAGAAGTGACGTTGGTGGATTTGACTCTGTCTCCTGGTACCAACAATCCCCAGGGAAAGCCCCCAAAGTCATGGTTTTTGATGTCAGTCATCGGCCCTCAGGTATCTCTAATCGCTTCTCTGGCTCCAAGTCCGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCACATTGAGGACGAGGGCGATTATTTCTGCTCTTCACTGACAGACAGAAGCCATCGCATATTCGGCGGCGGGACCAAGGTGACCGTTCTA

1503_H05 lambda light chain amino acid sequence: expressed protein withvariable region in bold.

(SEQ ID NO: 56) QSALTQPASVSGSPGQTITISCNGTRSDVGGFDSVSWYQQSPGKAPKVMVFDVSHRPSGISNRFSGSKSGNTASLTISGLHIEDEGDYFCSSLTDRSHRIFGGGTKVTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVT HEGSTVEKTVAPTECS

1503_H05 lambda light chain variable region amino acid sequence: (KabatCDRs underlined, Chothia CDRs in bold italics)

(SEQ ID NO: 57) QSALTQPASVSGSPGQTITISC

WYQQSPGKAPKVMVF

ISNRFS GSKSGNTASLTISGLHIEDEGDYFC

FGGGTKVTVL

1503_H05 lambda light chain Kabat CDRs:

CDR 1: NGTRSDVGGFDSVS (SEQ ID NO: 92) CDR 2: DVSHRPSG (SEQ ID NO: 95)CDR 3: SSLTDRSHRI (SEQ ID NO: 41)

1503_H05 lambda light chain Chothia CDRs:

CDR 1: NGTRSDVGGFDSVS (SEQ ID NO: 92) CDR 2: DVSHRPSG (SEQ ID NO: 95)CDR 3: SSLTDRSHRI (SEQ ID NO: 41)

1489_I13 gamma heavy chain nucleotide sequence: 1489_I13 γ3 codingsequence (variable region in bold)

(SEQ ID NO: 58) ATGGAGTTTGGGCTGAGCTGGGTTTTCCTCGCAACTCTGTTAAGAGTTGTGAAGTGTCAGGAACAACTGTTGGAGTCTGGGGGAGGCGTGGTCCAGCCGGGGGGGTCCCTGAGACTCTCCTGTTTAGCGTCTGGATTCACGTTTCACAAATATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGCCTGGAGTGGGTGGCACTCATCTCAGATGACGGAATGAGGAAATATCATTCAAACTCCATGTGGGGCCGAGTCACCATCTCCAGAGACAATTCCAAGAACACTCTTTATCTGCAATTCAGCAGCCTGAAAGTCGAAGACACGGCTATGTTCTTCTGTGCGAGAGAGGCTGGTGGGCCAATCTGGCATGACGACGTCAAATATTACGATTTTAATGACGGCTACTACAACTACCACTACATGGACGTCTGGGGCAAGGGGACCACGGTCACCGTCTCCTCAGCGTCGACCAAGGGCCCATCGGTCTTCCCTCTGGCACCATCATCCAAGTCGACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTC CGGGTAAATGA

1489_I13 gamma heavy chain variable region nucleotide sequence:

(SEQ ID NO: 134) CAGGAACAACTGTTGGAGTCTGGGGGAGGCGTGGTCCAGCCGGGGGGGTCCCTGAGACTCTCCTGTTTAGCGTCTGGATTCACGTTTCACAAATATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGCCTGGAGTGGGTGGCACTCATCTCAGATGACGGAATGAGGAAATATCATTCAAACTCCATGTGGGGCCGAGTCACCATCTCCAGAGACAATTCCAAGAACACTCTTTATCTGCAATTCAGCAGCCTGAAAGTCGAAGACACGGCTATGTTCTTCTGTGCGAGAGAGGCTGGTGGGCCAATCTGGCATGACGACGTCAAATATTACGATTTTAATGACGGCTACTACAACTACCACTACATGGACGTCTGGGGCAAGGGGACCACGGTCA CCGTCTCCTCA

1489_I13 gamma heavy chain amino acid sequence: expressed protein withvariable region in bold.

(SEQ ID NO: 59) QEQLLESGGGVVQPGGSLRLSCLASGFTFHKYGMHWVRQAPGKGLEWVALISDDGMRKYHSNSMWGRVTISRDNSKNTLYLQFSSLKVEDTAMFFCAREAGGPIWHDDVKYYDFNDGYYNYHYMDVWGKGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK

1489_I13 gamma heavy chain variable region amino acid sequence: (KabatCDRs underlined, Chothia CDRs in bold italics)

(SEQ ID NO: 60) QEQLLESGGGVVQPGGSLRLSCLA

WVRQAPGKGLEWVA

GRVT ISRDNSKNTLYLQFSSLKVEDTAMFFCAR

WGKGTTVTVSS

1489_I13 gamma heavy chain Kabat CDRs:

CDR 1: (SEQ ID NO: 88) SGFTFHKYGMH CDR 2: (SEQ ID NO: 98)LISDDGMRKYHSNSMW CDR 3: (SEQ ID NO: 6) EAGGPIWHDDVKYYDFNDGYYNYHYMDV

1489_I13 gamma heavy chain Chothia CDRs:

CDR 1: (SEQ ID NO: 88) SGFTFHKYGMH CDR 2: (SEQ ID NO: 98)LISDDGMRKYHSNSMW CDR 3: (SEQ ID NO: 6) EAGGPIWHDDVKYYDFNDGYYNYHYMDV

1489_I13 lambda light chain nucleotide sequence: 1489_I13 λ2 codingsequence (variable region in bold)

(SEQ ID NO: 61) ATGGCCTGGGCTCTGCTATTCCTCACCCTCTTCACTCAGGGCACAGGGTCCCGGGGCCAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGACGATCACCATCTCCTGCAATGGAACCAGCAGTGACGTTGGTGGATTTGACTCTGTCTCCTGGTATCAACAATCCCCAGGGAAAGCCCCCAAAGTCATGGTTTTTGATGTCAGTCATCGGCCCTCAGGTATCTCTAATCGCTTCTCTGGCTCCAAGTCCGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCACATTGAGGACGAGGGCGATTATTTCTGCTCTTCACTGACAGACAGAAGCCATCGCATATTCGGCGGCGGGACCAAGGTGACCGTTCTAGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTACCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAAAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAAT GTTCATAG

1489_I13 lambda light chain variable region nucleotide sequence:

(SEQ ID NO: 135) CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGACGATCACCATCTCCTGCAATGGAACCAGCAGTGACGTTGGTGGATTTGACTCTGTCTCCTGGTATCAACAATCCCCAGGGAAAGCCCCCAAAGTCATGGTTTTTGATGTCAGTCATCGGCCCTCAGGTATCTCTAATCGCTTCTCTGGCTCCAAGTCCGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCACATTGAGGACGAGGGCGATTATTTCTGCTCTTCACTGACAGACAGAAGCCATCGCATATTCGGCGGCGGGACCAAGGTGACCGTTCTA

1489_I13 lambda light chain amino acid sequence: expressed protein withvariable region in bold.

(SEQ ID NO: 14) QSALTQPASVSGSPGQTITISCNGTSSDVGGFDSVSWYQQSPGKAPKVMVFDVSHRPSGISNRFSGSKSGNTASLTISGLHIEDEGDYFCSSLTDRSHRIFGGGTKVTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVT HEGSTVEKTVAPTECS

1489_I13 lambda light chain variable region amino acid sequence: (KabatCDRs underlined, Chothia CDRs in bold italics).

(SEQ ID NO: 32) QSALTQPASVSGSPGQTITISC

WYQQSPGKAPKVMVF

ISNRFS GSKSGNTASLTISGLHIEDEGDYFC

FGGGTKVTVL

1489_I13 lambda light chain Kabat CDRs:

CDR 1: NGTSSDVGGFDSVS (SEQ ID NO: 97) CDR 2: DVSHRPSG (SEQ ID NO: 95)CDR 3: SSLTDRSHRI (SEQ ID NO: 41)

1489_I13 lambda light chain Chothia CDRs:

CDR 1: NGTSSDVGGFDSVS (SEQ ID NO: 97) CDR 2: DVSHRPSG (SEQ ID NO: 95)CDR 3: SSLTDRSHRI (SEQ ID NO: 41)

1480_I08 gamma heavy chain nucleotide sequence: 1480_I08 γ3 codingsequence (variable region in bold)

(SEQ ID NO: 64) ATGGAGTTTGGCTGAGCTGGGTTTTCCTCGCAACTCTGTTAAGAGTTGTGAAGTGTCAGGAACAACTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCGGGGGGGTCCCTGAGACTCTCCTGTTTAGCGTCTGGATTCACGTTTCACAAATATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGCCTGGAGTGGGTGGCACTCATCTCAGATGACGGAATGAGGAAATATCATTCAGACTCCATGTGGGGCCGAGTCACCATCTCCAGAGACAATTCCAAGAACACTCTTTATCTGCAATTCAGCAGCCTGAAAGTCGAAGACACGGCTATGTTCTTCTGTGCGAGAGAGGCTGGTGGGCCAATCTGGCATGACGACGTCAAATATTACGATTTTAATGACGGCTACTACAACTACCACTACATGGACGTCTGGGGCAAGGGGACCACGGTCACCGTCTCCTCAGCGTCGACCAAGGGCCCATCGGTCTTCCCTCTGGCACCATCATCCAAGTCGACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCC GGGTAAATGA

1480_I08 gamma heavy chain variable region nucleotide sequence:

(SEQ ID NO: 136) CAGGAACAACTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCGGGGGGGTCCCTGAGACTCTCCTGTTTAGCGTCTGGATTCACGTTTCACAAATATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGCCTGGAGTGGGTGGCACTCATCTCAGATGACGGAATGAGGAAATATCATTCAGACTCCATGTGGGGCCGAGTCACCATCTCCAGAGACAATTCCAAGAACACTCTTTATCTGCAATTCAGCAGCCTGAAAGTCGAAGACACGGCTATGTTCTTCTGTGCGAGAGAGGCTGGTGGGCCAATCTGGCATGACGACGTCAAATATTACGATTTTAATGACGGCTACTACAACTACCACTACATGGACGTCTGGGGCAAGGGGACCACGGTCA CCGTCTCCTCA

1480_I08 gamma heavy chain amino acid sequence: expressed protein withvariable region in bold.

(SEQ ID NO: 65) QEQLVESGGGVVQPGGSLRLSCLASGFTFHKYGMHWVRQAPGKGLEWVALISDDGMRKYHSDSMWGRVTISRDNSKNTLYLQFSSLKVEDTAMFFCAREAGGPIWHDDVKYYDFNDGYYNYHYMDVWGKGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK

1480_I08 gamma heavy chain variable region amino acid sequence: (KabatCDRs underlined, Chothia CDRs in bold italics)

(SEQ ID NO: 31) QEQLVESGGGVVQPGGSLRLSCLA

WVRQAPGKGLEW VA

GRVTISRDNSKNTLYLQFSS LKVEDTAMFFCAR

WG KGTTVTVSS

1480_I08 gamma heavy chain Kabat CDRs:

CDR 1: (SEQ ID NO: 88) SGFTFHKYGMH CDR 2: (SEQ ID NO: 89)LISDDGMRKYHSDSMW CDR 3: (SEQ ID NO: 6) EAGGPIWHDDVKYYDFNDGYYNYHYMDV

1480_I08 gamma heavy chain Chothia CDRs:

CDR 1: (SEQ ID NO: 88) SGFTFHKYGMH CDR 2: (SEQ ID NO: 89)LISDDGMRKYHSDSMW CDR 3: (SEQ ID NO: 6) EAGGPIWHDDVKYYDFNDGYYNYHYMDV

1480_I08 lambda light chain nucleotide sequence: 1480_I08□λ2 codingsequence (variable region in bold)

(SEQ ID NO: 67) ATGGCCTGGGCTCTGCTATTCGTCACCCTCCTCACTCAGGGCACAGGGTCCTGGGGCCAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGACGATCACCATCTCCTGCAATGGAACCAGCAGTGACGTTGGTGGATTTGACTCTGTCTCCTGGTATCAACAATCCCCAGGGAAAGCCCCCAAAGTCATGGTTTTTGATGTCAGTCATCGGCCCTCAGGTATCTCTAATCGCTTCTCTGGCTCCAAGTCCGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCACATTGAGGACGAGGGCGATTATTTCTGCTCTTCACTGACAGACAGAAGCCATCGCATATTCGGCGGCGGGACCAAGGTGACCGTTCTAGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTACCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAAAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAAT GTTCATAG

1480_I08 lambda light chain variable region nucleotide sequence:

(SEQ ID NO: 137) CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGACGATCACCATCTCCTGCAATGGAACCAGCAGTGACGTTGGTGGATTTGACTCTGTCTCCTGGTATCAACAATCCCCAGGGAAAGCCCCCAAAGTCATGGTTTTTGATGTCAGTCATCGGCCCTCAGGTATCTCTAATCGCTTCTCTGGCTCCAAGTCCGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCACATTGAGGACGAGGGCGATTATTTCTGCTCTTCACTGACAGACAGAAGCCATCGCATATTCGGCGGCGGGACCAAGGTGACCGTTCTA

1480_I08 lambda light chain amino acid sequence: expressed protein withvariable region in bold.

(SEQ ID NO: 14) QSALTQPASVSGSPGQTITISCNGTSSDVGGFDSVSWYQQSPGKAPKVMVFDVSHRPSGISNRFSGSKSGNTASLTISGLHIEDEGDYFCSSLTDRSHRIFGGGTKVTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVT HEGSTVEKTVAPTECS

1480_I08 lambda light chain variable region amino acid sequence: (KabatCDRs underlined, Chothia CDRs in bold italics)

(SEQ ID NO: 32) QSALTQPASVSGSPGQTITISC

WYQQSPGKAPKVM VF

ISNRFSGSKSGNTASLTISGLHIEDEGDYFC

F GGGTKVTVL

1480_I08 lambda light chain Kabat CDRs:

CDR 1: NGTSSDVGGFDSVS (SEQ ID NO: 97) CDR 2: DVSHRPSG (SEQ ID NO: 95)CDR 3: SSLTDRSHRI (SEQ ID NO: 41)1480_I08 lambda light chain Chothia CDRs:

CDR 1: NGTSSDVGGFDSVS (SEQ ID NO: 97) CDR 2: DVSHRPSG (SEQ ID NO: 95)CDR 3: SSLTDRSHRI (SEQ ID NO: 41)

The 1469_M23 (PG16) antibody includes a heavy chain variable region (SEQID NO: 139), encoded by the nucleic acid sequence shown in SEQ ID NO:128, and a light chain variable region (SEQ ID NO: 142) encoded by thenucleic acid sequence shown in SEQ ID NO: 129.

The heavy chain CDRs of the 1469_M23 (PG16) antibody have the followingsequences per Kabat and Chothia definitions: SGFTFHKYGMH (SEQ ID NO:88), LISDDGMRKYHSDSMW (SEQ ID NO: 89), and EAGGPIWHDDVKYYDFNDGYYNYHYMDV(SEQ ID NO: 6). The light chain CDRs of the 1469_M23 (PG16) antibodyhave the following sequences per Kabat and Chothia definitions:NGTRSDVGGFDSVS (SEQ ID NO: 92), DVSHRPSG (SEQ ID NO: 95), and SSLTDRSHRI(SEQ ID NO: 41).

The 1456_A12 (PG16) antibody includes a heavy chain variable region (SEQID NO: 47), encoded by the nucleic acid sequence shown in SEQ ID NO:130, and a light chain variable region (SEQ ID NO: 50) encoded by thenucleic acid sequence shown in SEQ ID NO: 131.

The heavy chain CDRs of the 1456_A12 (PG16) antibody have the followingsequences per Kabat and Chothia definitions: SGFTFHKYGMH (SEQ ID NO:88), LISDDGMRKYHSDSMW (SEQ ID NO: 89), and EAGGPIWHDDVKYYDFNDGYYNYHYMDV(SEQ ID NO: 6). The light chain CDRs of the 1456_A12 (PG16) antibodyhave the following sequences per Kabat and Chothia definitions:NGTSRDVGGFDSVS (SEQ ID NO: 93), DVSHRPSG (SEQ ID NO: 95), and SSLTDRSHRI(SEQ ID NO: 41).

The 1503_H05 (PG16) antibody includes a heavy chain variable region (SEQID NO: 53), encoded by the nucleic acid sequence shown in SEQ ID NO:132, and a light chain variable region (SEQ ID NO: 56) encoded by thenucleic acid sequence shown in SEQ ID NO: 133.

The heavy chain CDRs of the 1503_H05 (PG16) antibody have the followingsequences per Kabat and Chothia definitions: SGFTFHKYGMH (SEQ ID NO:88), LISDDGMRKYHSDSMW (SEQ ID NO: 89), and EAGGPIWHDDVKYYDFNDGYYNYHYMDV(SEQ ID NO: 6). The light chain CDRs of the 1503_H05 (PG16) antibodyhave the following sequences per Kabat and Chothia definitions:NGTRSDVGGFDSVS (SEQ ID NO: 92), DVSHRPSG (SEQ ID NO: 95), and SSLTDRSHRI(SEQ ID NO: 41).

The 1489_I13 (PG16) antibody includes a heavy chain variable region (SEQID NO: 59), encoded by the nucleic acid sequence shown in SEQ ID NO:134, and a light chain variable region (SEQ ID NO: 14) encoded by thenucleic acid sequence shown in SEQ ID NO: 135.

The heavy chain CDRs of the 1489_I13 (PG16) antibody have the followingsequences per Kabat and Chothia definitions: SGFTFHKYGMH (SEQ ID NO:88), LISDDGMRKYHSNSMW (SEQ ID NO: 98), and EAGGPIWHDDVKYYDFNDGYYNYHYMDV(SEQ ID NO: 6). The light chain CDRs of the 1489_I13 (PG16) antibodyhave the following sequences per Kabat and Chothia definitions:NGTSSDVGGFDSVS (SEQ ID NO: 97), DVSHRPSG (SEQ ID NO: 95), and SSLTDRSHRI(SEQ ID NO: 41).

The 1480_I08 (PG16) antibody includes a heavy chain variable region (SEQID NO: 65), encoded by the nucleic acid sequence shown in SEQ ID NO:136, and a light chain variable region (SEQ ID NO: 14) encoded by thenucleic acid sequence shown in SEQ ID NO: 137.

The heavy chain CDRs of the 1480_I08 (PG16) antibody have the followingsequences per Kabat and Chothia definitions: SGFTFHKYGMH (SEQ ID NO:88), LISDDGMRKYHSDSMW (SEQ ID NO: 89), and EAGGPIWHDDVKYYDFNDGYYNYHYMDV(SEQ ID NO: 6). The light chain CDRs of the 1480_I08 (PG16) antibodyhave the following sequences per Kabat and Chothia definitions:NGTSSDVGGFDSVS (SEQ ID NO: 97), DVSHRPSG (SEQ ID NO: 95), and SSLTDRSHRI(SEQ ID NO: 41).

In one aspect, an antibody according to the invention contains a heavychain having the amino acid sequence of SEQ ID NOs: 12, 16, 20, 24, 28,139, 47, 53, 59, or 65 and a light chain having the amino acid sequenceof SEQ ID NOs: 14, 18, 22, 26, 30, 142, 50, or 56. Alternatively, anantibody according to the invention contains a heavy chain variableregion having the amino acid sequence of SEQ ID NOs: 31, 33, 35, 37, 39,140, 48, 54, or 60 and a light chain variable region having the aminoacid sequence of SEQ ID NOs: 32, 34, 36, 38, 40, 96, 51, or 57.

In another aspect, an antibody according to the invention contains aheavy chain having the amino acid sequence encoded by the nucleic acidsequence of SEQ ID NOs: 11, 15, 19, 23, 27, 138, 46, 52, 58, or 64 and alight chain having the amino acid sequence encoded by the nucleic acidsequence of SEQ ID NOs: 13, 17, 21, 25, 29, 141, 49, 55, 61, or 67.Alternatively, an antibody according to the invention contains a heavychain variable region having the amino acid sequence encoded by thenucleic acid sequence of SEQ ID NOs: 99, 101, 109, 115, 122, 128, 130,132, 134, or 136 and a light chain variable region having the amino acidsequence encoded by the nucleic acid sequence of SEQ ID NOs: 100, 106,112, 119, 125, 129, 131, 133, 135, or 137. Furthermore, an antibodyaccording to the invention contains a heavy chain having the amino acidsequence encoded by a nucleic acid sequence of SEQ ID NOs: 11, 15, 19,23, 27, 138, 46, 52, 58, or 64, which contains a silent or degeneratemutation, and a light chain having the amino acid sequence encoded bythe nucleic acid sequence of SEQ ID NOs: 13, 17, 21, 25, 29, 141, 49,55, 61, or 67, which contains a silent or degenerate mutation. Silentand degenerate mutations alter the nucleic acid sequence, but do notalter the resultant amino acid sequence.

Preferably the three heavy chain CDRs include an amino acid sequence ofat least 90%, 92%, 95%, 97%, 98%, 99%, or more identical to the aminoacid sequence of SGFTFHKYGMH (SEQ ID NO: 88), LISDDGMRKYHSDSMW (SEQ IDNO: 89), EAGGPIWHDDVKYYDFNDGYYNYHYMDV (SEQ ID NO: 6), SGGTFSSYAFT (SEQID NO: 104), MVTPIFGEAKYSQRFE (SEQ ID NO: 105), RAVPIATDNWLDP (SEQ IDNO: 102), SGGAFSSYAFS (SEQ ID NO: 110), MITPVFGETKYAPRFQ (SEQ ID NO:111), SGYSFIDYYLH (SEQ ID NO: 116), LIDPENGEARYAEKFQ (SEQ ID NO: 117),AVGADSGSWFDP (SEQ ID NO: 118), SGFDFSRQGMH (SEQ ID NO: 123),FIKYDGSEKYHADSVW (SEQ ID NO: 124), EAGGPDYRNGYNYYDFYDGYYNYHYMDV (SEQ IDNO: 7), LISDDGMRKYHSNSMW (SEQ ID NO: 98) (as determined by the Kabatmethod) or SGFTFHKYGMH (SEQ ID NO: 88), LISDDGMRKYHSDSMW (SEQ ID NO:89), EAGGPIWHDDVKYYDFNDGYYNYHYMDV (SEQ ID NO: 6), SGGTFSSYAFT (SEQ IDNO: 104), MVTPIFGEAKYSQRFE (SEQ ID NO: 105), RRAVPIATDNWLDP (SEQ ID NO:103), SGGAFSSYAFS (SEQ ID NO: 110), MITPVFGETKYAPRFQ (SEQ ID NO: 111),SGYSFIDYYLH (SEQ ID NO: 116), LIDPENGEARYAEKFQ (SEQ ID NO: 117),AVGADSGSWFDP (SEQ ID NO: 118), SGFDFSRQGMH (SEQ ID NO: 123),FIKYDGSEKYHADSVW (SEQ ID NO: 124), EAGGPDYRNGYNYYDFYDGYYNYHYMDV (SEQ IDNO: 7), LISDDGMRKYHSNSMW (SEQ ID NO: 98) (as determined by the Chothiamethod) and a light chain with three CDRs that include an amino acidsequence of at least 90%, 92%, 95%, 97%, 98%, 99%, or more identical tothe amino acid sequence of NGTSSDVGGFDSVS (SEQ ID NO: 97), DVSHRPSG (SEQID NO: 95), SSLTDRSHRI (SEQ ID NO: 41), RASQTINNYLN (SEQ ID NO: 107).GASNLQNG (SEQ ID NO: 108), QQSFSTPRT (SEQ ID NO: 42), RASQTIHTYL (SEQ IDNO: 113), GASTLQSG (SEQ ID NO: 114), QQSYSTPRT (SEQ ID NO: 43),SGSKLGDKYVS (SEQ ID NO: 120), ENDRRPSG (SEQ ID NO: 121), QAWETIITITFVF(SEQ ID NO: 44), NGTSNDVGGYESVS (SEQ ID NO: 126), DVSKRPSG (SEQ ID NO:127), KSLTSTRRRV (SEQ ID NO: 45), NGTRSDVGGFDSVS (SEQ ID NO: 92),NGTSRDVGGFDSVS (SEQ ID NO: 93) (as determined by the Kabat method) orNGTSSDVGGFDSVS (SEQ ID NO: 97), DVSHRPSG (SEQ ID NO: 95), SSLTDRSHRI(SEQ ID NO: 41), RASQTINNYLN (SEQ ID NO: 107), GASNLQNG (SEQ ID NO:108), QQSFSTPRT (SEQ ID NO: 42), RASQTIHTYL (SEQ ID NO: 113), GASTLQSG(SEQ ID NO: 114), QQSYSTPRT (SEQ ID NO: 43), SGSKLGDKYVS (SEQ ID NO:120), ENDRRPSG (SEQ ID NO: 121), QAWETTTITFVF (SEQ ID NO: 44),NGTSNDVGGYESVS (SEQ ID NO: 126), DVSKRPSG (SEQ ID NO: 127), KSLTSTRRRV(SEQ ID NO: 45), NGTRSDVGGFDSVS (SEQ ID NO: 92), NGTSRDVGGFDSVS (SEQ IDNO: 93) (as determined by the Chothia method).

The heavy chain of the anti-HIV monoclonal antibody is derived from agerm line variable (V) gene such as, for example, the IGHV1 or IGHV3germline gene.

The anti-HIV antibodies of the invention include a variable heavy chain(V_(H)) region encoded by a human IGHV1 or IGHV3 germline gene sequence.IGHV1 germline gene sequences are shown, e.g., in Accession numbersL22582, X27506, X92340, M83132, X67905, L22583, Z29978, Z14309, Z14307,Z14300, Z14296, and Z14301. IGHV3 germline gene sequences are shown,e.g., in Accession numbers AB019439, M99665, M77305, M77335, and M77334.The anti-HIV antibodies of the invention include a V_(H) region that isencoded by a nucleic acid sequence that is at least 80% homologous tothe IGHV1 or IGHV3 germline gene sequence. Preferably, the nucleic acidsequence is at least 90%, 95%, 96%, 97% homologous to the IGHV1 or IGHV3germline gene sequence, and more preferably, at least 98%, 99%homologous to the IGHV1 or IGHV3 germline gene sequence. The V_(H)region of the anti-HIV antibody is at least 80% homologous to the aminoacid sequence of the V_(H) region encoded by the IGHV1 or IGHV3 V_(H)germline gene sequence. Preferably, the amino acid sequence of V_(H)region of the anti-HIV antibody is at least 90%, 95%, 96%, 97%homologous to the amino acid sequence encoded by the IGHV1 or IGHV3germline gene sequence, and more preferably, at least 98%, 99%homologous to the sequence encoded by the IGHV1 or IGHV3 germline genesequence.

The light chain of the anti-HIV monoclonal antibody is derived from agerm line variable (V) gene such as, for example, the IGLV2, IGLV3 orIGKV1 germline gene.

The anti-HIV antibodies of the invention also include a variable lightchain (V_(L)) region encoded by a human IGLV2, IGLV3 or IGKV1 germlinegene sequence. A human IGLV2 V_(L) germline gene sequence is shown,e.g., Accession numbers Z73664, L27822, Y12412, and Y12413. A humanIGLV3 V_(L) germline gene sequence is shown, e.g., Accession numberX57826.

A human IGKV1 V_(L) germline gene sequence is shown, e.g., Accessionnumbers AF306358, AF490911, L12062, L12064, L12065, L12066, L12068,L12072, L12075, L12076, L12079, L12080, L12081, L12082, L12083, L12084,L12085, L12086, 12088, L12091, L12093. L12101, L12106, L12108, L12110,L12112, M95721, M95722, M95723, X73855, X73860, X98972, X98973, Z15073,Z15074, Z15075, Z15077, Z15079, Z15081. Alternatively, the anti-HIVantibodies include a V_(L) region that is encoded by a nucleic acidsequence that is at least 80% homologous to the IGLV2, IGLV3 or IGKV1germline gene sequence. Preferably, the nucleic acid sequence is atleast 90%, 95%, 96%, 97% homologous to the IGLV2, IGLV3 or IGKV1germline gene sequence, and more preferably, at least 98%, 99%homologous to the IGLV2, IGLV3 or IGKV1 germline gene sequence. TheV_(L) region of the anti-CMV antibody is at least 80% homologous to theamino acid sequence of the V_(L) region encoded the IGLV2, IGLV3 orIGKV1 germline gene sequence. Preferably, the amino acid sequence ofV_(L) region of the anti-HIV antibody is at least 90%, 95%, 96%, 97%homologous to the amino acid sequence encoded by the IGLV2, IGLV3 orIGKV1 germline gene sequence, and more preferably, at least 98%, 99%homologous to the sequence encoded by the IGLV2, IGLV3 or IGKV1 germlinegene sequence.

TABLE 7Alignment of heavy chain coding sequences of the variable domain of 1443C16 sister clones to 1443 C16 and 1496 C09. Kabat CDR sequences for the PG16 sister clonesare highlighted in boxes.

TABLE 8Alignment of light chain coding sequences of the variable domain of 1443 C16sister clones to 1443 C16 and 1406 C09. Kabat CDR sequences for the PG16 sister clones arehighlighted in boxes.

TABLE 9Alignment of heavy chain protein sequences of the variable domain of 1443C16 sister clones to 1443 C16 and 1496 C09. Kabat CDR sequences for the PG16 sister clonesare highlighted in boxes.

TABLE 10Alignment of light chain protein sequences of the variable domain of 1443C16 sister clones to 1443 C16 and 1406 C09. Kabat CDR sequences for the PG16 sister clonesare highlighted in black boxes.

TABLE 11 Consensus nucleotide sequences of Kabat CDRs of heavy chains of1443 PG16 sister clones. CDR1: 1443 C16TCTGGATTCACGTTTCACAAATATGGCATGCAC (SEQ ID NO: 68) 1469 M23TCTGGATTCACCTTTCACAAATATGGCATGCAC (SEQ ID NO: 69) 1456 A12TCTGGATTCACGTTTCACAAATATGGCATGCAC (SEQ ID NO: 68) 1503 H05TCTGGATTCACCTTTCACAAATATGGCATGCAC (SEQ ID NO: 70) 1489 I13TCTGGATTCACGTTTCACAAATATGGCATGCAC (SEQ ID NO: 68) 1480 I08TCTGGATTCACGTTTCACAAATATGGCATGCAC (SEQ ID NO: 68) Consensus*TCTGGATTCACXTTTCACAAATATGGCATGCAC (SEQ ID NO: 71) Variation1TCTGGATTCACGTTTCACAAATATGGCATGCAC (SEQ ID NO: 68) Variation2TCTGGATTCACCTTTCACAAATATGGCATGCAC (SEQ ID NO: 70) *Wherein X is C or G.CDR2: 1443 C16 CTCATCTCAGATGACGGAATGAGGAAATATCATTCAGACTCCATGTGG (SEQ IDNO: 72) 1469 M23 CTCATCTCAGATGACGGAATGAGGAAATATCATTCAGACTCCATGTGG (SEQID NO: 72) 1456 A12 CTCATCTCAGATGACGGAATGAGGAAATATCATTCAGACTCCATGTGG(SEQ ID NO: 72) 1503 H05CTCATCTCAGATGACGGAATGAGGAAATATCATTCAGACTCCATGTGG (SEQ ID NO: 72) 1489I13 CTCATCTCAGATGACGGAATGAGGAAATATCATTCAAACTCCATGTGG (SEQ ID NO: 73)1480 I08 CTCATCTCAGATGACGGAATGAGGAAATATCATTCAGACTCCATGTGG (SEQ ID NO:72) Consensus* CTCATCTCAGATGACGGAATGAGGAAATATCATTCAXACTCCATGTGG (SEQ IDNO: 74) Variation1 CTCATCTCAGATGACGGAATGAGGAAATATCATTCAGACTCCATGTGG (SEQID NO: 72) Variation2 CTCATCTCAGATGACGGAATGAGGAAATATCATTCAAACTCCATGTGG(SEQ ID NO: 73) *Wherein X is A or G. CDR3: 1443 C16 (SEQ ID NO: 75)GAGGCTGGTGGGCCAATCTGGCATGACGACGTCAAATATTACGATTTTAATGACGGCTACTACAACTACCACTACATGGACGTC1469 M23 (SEQ ID NO: 75)GAGGCTGGTGGGCCAATCTGGCATGACGACGTCAAATATTACGATTTTAATGACGGCTACTACAACTACCACTACATGGACGTC1456 A12 (SEQ ID NO: 77)GAGGCCGGTGGGCCAATCTGGCATGACGACGTCAAATATTACGATTTTAATGACGGCTACTACAACTATCACTACATGGACGTC1503 H05 (SEQ ID NO: 79)GAGGCTGGTGGGCCAATCTGGCATGACGACGTCAAATATTACGATTTTAATGACGGCTACTACAATTACCACTACATGGACGTC1489 I13 (SEQ ID NO: 75)GAGGCTGGTGGGCCAATCTGGCATGACGACGTCAAATATTACGATTTTAATGACGGCTACTACAACTACCACTACATGGACGTC1480 I08 (SEQ ID NO: 75)GAGGCTGGTGGGCCAATCTGGCATGACGACGTCAAATATTACGATTTTAATGACGGCTACTACAACTACCACTACATGGACGTCConsensus (SEQ ID NO: 76)GAGGCXGGTGGGCCAATCTGGCATGACGACGTCAAATATTACGATTTTAATGACGGCTACTACAACTATCACTACATGGACGTCVariation1 (SEQ ID NO: 78)GAGGCGGGTGGGCCAATCTGGCATGACGACGTCAAATATTACGATTTTAATGACGGCTACTACAACTATCACTACATGGACGTCVariation2 (SEQ ID NO: 77)GAGGCCGGTGGGCCAATCTGGCATGACGACGTCAAATATTACGATTTTAATGACGGCTACTACAACTATCACTACATGGACGTC*Wherein X is T, C or G.

TABLE 12 Consensus nucleotide sequences of Kabat CDRs of light chains of1443 PG16 sister clones. CDR1: 1443 C16AATGGAACCAGCAGTGACGTTGGTGGATTTGACTCTGTCTCC (SEQ ID NO: 80) 1469 M23AATGGAACCAGAAGTGACGTTGGTGGATTTGACTCTGTCTCC (SEQ ID NO: 82) 1456 A12AATGGAACCAGCCGTGACGTTGGTGGATTTGACTCTGTCTCC (SEQ ID NO: 83) 1503 H05AATGGAACCAGAAGTGACGTTGGTGGATTTGACTCTGTCTCC (SEQ ID NO: 82) 1489 I13AATGGAACCAGCAGTGACGTTGGTGGATTTGACTCTGTCTCC (SEQ ID NO: 80) 1480 I08AATGGAACCAGCAGTGACGTTGGTGGATTTGACTCTGTCTCC (SEQ ID NO: 80) Consensus*AATGGAACCAGX ₁ X ₂GTGACGTTGGTGGATTTGACTCTGTCTCC (SEQ ID NO: 81)Variation1 AATGGAACCAGCAGTGACGTTGGTGGATTTGACTCTGTCTCC (SEQ ID NO: 80)Variation2 AATGGAACCAGAAGTGACGTTGGTGGATTTGACTCTGTCTCC (SEQ ID NO: 82)Variation2 AATGGAACCAGCCGTGACGTTGGTGGATTTGACTCTGTCTCC (SEQ ID NO: 83)*Wherein X₁ is C or A. Wherein X₂ is C or A. CDR2: 1443 C16GATGTCAGTCATCGGCCCTCAGGT (SEQ ID NO: 84) 1469 M23GATGTCAGTCATCGGCCCTCAGGT (SEQ ID NO: 84) 1456 A12GATGTCAGTCATCGGCCCTCAGGT (SEQ ID NO: 84) 1503 H05GATGTCAGTCATCGGCCCTCAGGT (SEQ ID NO: 84) 1489 I13GATGTCAGTCATCGGCCCTCAGGT (SEQ ID NO: 84) 1480 I08GATGTCAGTCATCGGCCCTCAGGT (SEQ ID NO: 84) ConsensusGATGTCAGTCATCGGCCCTCAGGT (SEQ ID NO: 84) CDR3: 1443 C16TCTTCACTGACAGACAGAAGCCATCGCATA (SEQ ID NO: 85) 1469 M23TCTTCACTGACAGACAGAAGCCATCGCATA (SEQ ID NO: 85) 1456 A12TCTTCATTGACAGACAGAAGCCATCGCATA (SEQ ID NO: 86) 1503 H05TCTTCACTGACAGACAGAAGCCATCGCATA (SEQ ID NO: 85) 1489 I13TCTTCACTGACAGACAGAAGCCATCGCATA (SEQ ID NO: 85) 1480 I08TCTTCACTGACAGACAGAAGCCATCGCATA (SEQ ID NO: 85) Consensus*TCTTCAXTGACAGACAGAAGCCATCGCATA (SEQ ID NO: 87) Variation1TCTTCACTGACAGACAGAAGCCATCGCATA (SEQ ID NO: 85) Variation2TCTTCATTGACAGACAGAAGCCATCGCATA (SEQ ID NO: 86) *Wherein X is C or T.

TABLE 13 Consensus protein sequences of Kabat CDRs of Heavy chains of1443 PG16 sister clones. CDR1: 1443 C16 SGFTFHKYGMH (SEQ ID NO: 88) 1469M23 SGFTFHKYGMH (SEQ ID NO: 88) 1456 A12 SGFTFHKYGMH (SEQ ID NO: 88)1503 H05 SGFTFHKYGMH (SEQ ID NO: 88) 1489 I13 SGFTFHKYGMH (SEQ ID NO:88) 1480 I08 SGFTFHKYGMH (SEQ ID NO: 88) Consensus SGFTFHKYGMH (SEQ IDNO: 88) CDR2: 1443 C16 LISDDGMRKYHSDSMW (SEQ ID NO: 89) 1469 M23LISDDGMRKYHSDSMW (SEQ ID NO: 89) 1456 A12 LISDDGMRKYHSDSMW (SEQ ID NO:89) 1503 H05 LISDDGMRKYHSDSMW (SEQ ID NO: 89) 1489 I13 LISDDGMRKYHSNSMW(SEQ ID NO: 98) 1480 I08 LISDDGMRKYHSDSMW (SEQ ID NO: 89) Consensus*LISDDGMRKYHSXSMW (SEQ ID NO: 91) Variation1 LISDDGMRKYHSDSMW (SEQ ID NO:89) Variation2 LISDDGMRKYHSNSMW (SEQ ID NO: 98) *Wherein X is D or N, orwherein X is an amino acid with similar physical properties to either Dor N. CDR3: 1443 C16 EAGGPIWHDDVKYYDFNDGYYNYHYMDV (SEQ ID NO: 6) 1469M23 EAGGPIWHDDVKYYDFNDGYYNYHYMDV (SEQ ID NO: 6) 1456 A12EAGGPIWHDDVKYYDFNDGYYNYHYMDV (SEQ ID NO: 6) 1503 H05EAGGPIWHDDVKYYDFNDGYYNYHYMDV (SEQ ID NO: 6) 1489 I13EAGGPIWHDDVKYYDFNDGYYNYHYMDV (SEQ ID NO: 6) 1480 I08EAGGPIWHDDVKYYDFNDGYYNYHYMDV (SEQ ID NO: 6) ConsensusEAGGPIWHDDVKYYDFNDGYYNYHYMDV (SEQ ID NO: 6)

TABLE 14 Consensus protein sequences of Kabat CDRs of light chains of1443 PG16 sister clones. CDR1: 1443 C16 NGTSSDVGGFDSVS (SEQ ID NO: 97)1469 M23 NGTRSDVGGFDSVS (SEQ ID NO: 92) 1456 A12 NGTSRDVGGFDSVS (SEQ IDNO: 93) 1503 H05 NGTRSDVGGFDSVS (SEQ ID NO: 92) 1489 I13 NGTSSDVGGFDSVS(SEQ ID NO: 97) 1480 I08 NGTSSDVGGFDSVS (SEQ ID NO: 97) Consensus* NGTX₁ X ₂DVGGFDSVS (SEQ ID NO: 94) Variation1 NGTSSDVGGFDSVS (SEQ ID NO: 97)Variation2 NGTRSDVGGFDSVS (SEQ ID NO: 92) Variation3 NGTSRDVGGFDSVS (SEQID NO: 93) *Wherein X₁ is S or R, or wherein X₁ is an amino acid withsimilar physical properties to either S or R. Wherein X₂ is S or R, orwherein X₂ is an amino acid with similar physical properties to either Sor R. CDR2: 1443 C16 DVSHRPSG (SEQ ID NO: 95) 1469 M23 DVSHRPSG (SEQ IDNO: 95) 1456 A12 DVSHRPSG (SEQ ID NO: 95) 1503 H05 DVSHRPSG (SEQ ID NO:95) 1489 I13 DVSHRPSG (SEQ ID NO: 95) 1408 I08 DVSHRPSG (SEQ ID NO: 95)Consensus DVSHRPSG (SEQ ID NO: 95) CDR3: 1443 C16 SSLTDRSHRI (SEQ ID NO:41) 1469 M23 SSLTDRSHRI (SEQ ID NO: 41) 1456 A12 SSLTDRSHRI (SEQ ID NO:41) 1503 H05 SSLTDRSHRI (SEQ ID NO: 41) 1489 I13 SSLTDRSHRI (SEQ ID NO:41) 1480 I08 SSLTDRSHRI (SEQ ID NO: 41) Consensus SSLTDRSHRI (SEQ ID NO:41)

Monoclonal and recombinant antibodies are particularly useful inidentification and purification of the individual polypeptides or otherantigens against which they are directed. The antibodies of theinvention have additional utility in that they may be employed asreagents in immunoassays, radioimmunoassays (RIA) or enzyme-linkedimmunosorbent assays (ELISA). In these applications, the antibodies canbe labeled with an analytically-detectable reagent such as aradioisotope, a fluorescent molecule or an enzyme. The antibodies mayalso be used for the molecular identification and characterization(epitope mapping) of antigens.

As mentioned above, the antibodies of the invention can be used to mapthe epitopes to which they bind. Applicants have discovered that theantibodies 1496_C09 (PG9), 1443_C16 (PG16), 1456_P20 (PG20), 1460_G14(PGG14), 1495_C14 (PGC14), 1469_M23 (PG16), 1456_A12 (PG16), 1503_H05(PG16), 1489_I13 (PG16), and 1080_I08 (PG16) neutralize HIV. Althoughthe Applicant does not wish to be bound by this theory, it is postulatedthat the antibodies 1496_C09 (PG9), 1443_C16 (PG16), 1456_P20 (PG20),1460_G14 (PGG14), 1495_C14 (PGC14), 1469_M23 (PG16), 1456_A12 (PG16),1503_H05 (PG16), 1489_I13 (PG16), and/or 1080_(—)108 (PG16) bind to oneor more conformational epitopes formed by HIV1-encoded proteins.

Neutralization activity of human monoclonal antibodies was testedagainst HIV-1 strains SF162 and JR-CSF. HIV-1 strains SF162 and JR-CSFboth belong to HIV clade B. Each clonal monoclonal antibody was screenedfor neutralization activity and for anti-gp120, anti-gp41 and total IgGin quantitative ELISA. For the monoclonal antibodies 1456_P20, 1495_C14,and 1460_G14 anti-gp120 antigen-specific binding was detected.Neutralizing activity against SF162, but not JR-CSF was detected for1456_P20 (PG20), 1495_C14 (PGC14), and 1460_G14 (PGG14). For the twomonoclonal antibody preparations that did not show binding to gp120 inthe ELISA assay, 1443_C16 (PG16) and 1496_C09 (PG9), high quantities ofhuman IgG were determined to be present in the assay. However, 1443_C16(PG16) and 1496_C09 (PG9) both were found to exhibit neutralizingactivity against HIV-1 strain JR-CSF, but not against strain SF162.1443_C16 (PG16) and 1496_C09 (PG9) also were found to lack gp41 bindingactivity in the ELISA assay.

The epitopes recognized by these antibodies may have a number of uses.The epitopes and mimotopes in purified or synthetic form can be used toraise immune responses (i.e. as a vaccine, or for the production ofantibodies for other uses) or for screening patient serum for antibodiesthat immunoreact with the epitopes or mimotopes. Preferably, such anepitope or mimotope, or antigen comprising such an epitope or mimotopeis used as a vaccine for raising an immune response. The antibodies ofthe invention can also be used in a method to monitor the quality ofvaccines in particular to check that the antigen in a vaccine containsthe correct immunogenic epitope in the correct conformation.

The epitopes may also be useful in screening for ligands that bind tosaid epitopes. Such ligands preferably block the epitopes and thusprevent infection. Such ligands are encompassed within the scope of theinvention.

Standard techniques of molecular biology may be used to prepare DNAsequences coding for the antibodies or fragments of the antibodies ofthe present invention. Desired DNA sequences may be synthesizedcompletely or in part using oligonucleotide synthesis techniques.Site-directed mutagenesis and polymerase chain reaction (PCR) techniquesmay be used as appropriate.

Any suitable host cell/vector system may be used for expression of theDNA sequences encoding the antibody molecules of the present inventionor fragments thereof. Bacterial, for example E. coli, and othermicrobial systems may be used, in part, for expression of antibodyfragments such as Fab and F(ab′)₂ fragments, and especially Fv fragmentsand single chain antibody fragments, for example, single chain Fvs.Eukaryotic, e.g. mammalian, host cell expression systems may be used forproduction of larger antibody molecules, including complete antibodymolecules. Suitable mammalian host cells include CHO, HEK293T, PER.C6,myeloma or hybridoma cells.

The present invention also provides a process for the production of anantibody molecule according to the present invention comprisingculturing a host cell comprising a vector of the present invention underconditions suitable for leading to expression of protein from DNAencoding the antibody molecule of the present invention, and isolatingthe antibody molecule. The antibody molecule may comprise only a heavyor light chain polypeptide, in which case only a heavy chain or lightchain polypeptide coding sequence needs to be used to transfect the hostcells. For production of products comprising both heavy and lightchains, the cell line may be transfected with two vectors, a firstvector encoding a light chain polypeptide and a second vector encoding aheavy chain polypeptide. Alternatively, a single vector may be used, thevector including sequences encoding light chain and heavy chainpolypeptides.

Alternatively, antibodies according to the invention may be produced byi) expressing a nucleic acid sequence according to the invention in acell, and ii) isolating the expressed antibody product. Additionally,the method may include iii) purifying the antibody. Transformed B cellsare screened for those producing antibodies of the desired antigenspecificity, and individual B cell clones can then be produced from thepositive cells. The screening step may be carried out by ELISA, bystaining of tissues or cells (including transfected cells), aneutralization assay or one of a number of other methods known in theart for identifying desired antigen specificity. The assay may select onthe basis of simple antigen recognition, or may select on the additionalbasis of a desired function e.g. to select neutralizing antibodiesrather than just antigen-binding antibodies, to select antibodies thatcan change characteristics of targeted cells, such as their signalingcascades, their shape, their growth rate, their capability ofinfluencing other cells, their response to the influence by other cellsor by other reagents or by a change in conditions, their differentiationstatus, etc.

The cloning step for separating individual clones from the mixture ofpositive cells may be carried out using limiting dilution,micromanipulation, single cell deposition by cell sorting or anothermethod known in the art. Preferably the cloning is carried out usinglimiting dilution.

The immortalized B cell clones of the invention can be used in variousways e.g. as a source of monoclonal antibodies, as a source of nucleicacid (DNA or mRNA) encoding a monoclonal antibody of interest, forresearch, etc.

Unless otherwise defined, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Generally,nomenclatures utilized in connection with, and techniques of, cell andtissue culture, molecular biology, and protein and oligo- orpolynucleotide chemistry and hybridization described herein are thosewell known and commonly used in the art. Standard techniques are usedfor recombinant DNA, oligonucleotide synthesis, and tissue culture andtransformation (e.g., electroporation, lipofection). Enzymatic reactionsand purification techniques are performed according to manufacturer'sspecifications or as commonly accomplished in the art or as describedherein. The practice of the present invention will employ, unlessindicated specifically to the contrary, conventional methods ofvirology, immunology, microbiology, molecular biology and recombinantDNA techniques within the skill of the art, many of which are describedbelow for the purpose of illustration. Such techniques are explainedfully in the literature. See, e.g., Sambrook, et al. Molecular Cloning:A Laboratory Manual (2nd Edition, 1989); Maniatis et al. MolecularCloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach,vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed.,1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985);Transcription and Translation (B. Hames & S. Higgins, eds., 1984);Animal Cell Culture (R. Freshney, ed., 1986); Perbal, A Practical Guideto Molecular Cloning (1984). The nomenclatures utilized in connectionwith, and the laboratory procedures and techniques of, analyticalchemistry, synthetic organic chemistry, and medicinal and pharmaceuticalchemistry described herein are those well known and commonly used in theart. Standard techniques are used for chemical syntheses, chemicalanalyses, pharmaceutical preparation, formulation, and delivery, andtreatment of patients.

The following definitions are useful in understanding the presentinvention:

The term “antibody” (Ab) as used herein includes monoclonal antibodies,polyclonal antibodies, multispecific antibodies (e.g., bispecificantibodies), and antibody fragments, as long as they exhibit the desiredbiological activity. The term “immunoglobulin” (Ig) is usedinterchangeably with “antibody” herein.

A “neutralizing antibody” may inhibit the entry of HIV-1 virus forexample SF162 and/or JR-CSF with a neutralization index >1.5 or >2.0.(Kostrikis L G et al. J Virol. 1996; 70(1): 445-458.) By “broad andpotent neutralizing antibodies” are meant antibodies that neutralizemore than one HIV-1 virus species (from diverse clades and differentstrains within a clade) in a neutralization assay. A broad neutralizingantibody may neutralize at least 2, 3, 4, 5, 6, 7, 8, 9 or moredifferent strains of HIV-1, the strains belonging to the same ordifferent clades. A broad neutralizing antibody may neutralize multipleHIV-1 species belonging to at least 2, 3, 4, 5, or 6 different clades.The inhibitory concentration of the monoclonal antibody may be less thanabout 25 mg/ml to neutralize about 50% of the input virus in theneutralization assay.

An “isolated antibody” is one that has been separated and/or recoveredfrom a component of its natural environment. Contaminant components ofits natural environment are materials that would interfere withdiagnostic or therapeutic uses for the antibody, and may includeenzymes, hormones, and other proteinaceous or nonproteinaceous solutes.In preferred embodiments, the antibody is purified: (1) to greater than95% by weight of antibody as determined by the Lowry method, and mostpreferably more than 99% by weight; (2) to a degree sufficient to obtainat least 15 residues of N-terminal or internal amino acid sequence byuse of a spinning cup sequenator; or (3) to homogeneity by SDS-PAGEunder reducing or non-reducing conditions using Coomassie blue or,preferably, silver stain. Isolated antibody includes the antibody insitu within recombinant cells since at least one component of theantibody's natural environment will not be present. Ordinarily, however,isolated antibody will be prepared by at least one purification step.

The basic four-chain antibody unit is a heterotetrameric glycoproteincomposed of two identical light (L) chains and two identical heavy (H)chains. An IgM antibody consists of 5 basic heterotetramer units alongwith an additional polypeptide called J chain, and therefore contain 10antigen binding sites, while secreted IgA antibodies can polymerize toform polyvalent assemblages comprising 2-5 of the basic 4-chain unitsalong with J chain. In the case of IgGs, the 4-chain unit is generallyabout 150,000 daltons. Each L chain is linked to an H chain by onecovalent disulfide bond, while the two H chains are linked to each otherby one or more disulfide bonds depending on the H chain isotype. Each Hand L chain also has regularly spaced intrachain disulfide bridges. EachH chain has at the N-terminus, a variable region (V_(H)) followed bythree constant domains (C_(H)) for each of the α and γ chains and fourC_(H) domains for μ and ε isotypes. Each L chain has at the N-terminus,a variable region (V_(L)) followed by a constant domain (C_(L)) at itsother end. The V_(L) is aligned with the V_(H) and the C_(L) is alignedwith the first constant domain of the heavy chain (C_(H)1). Particularamino acid residues are believed to form an interface between the lightchain and heavy chain variable regions. The pairing of a V_(H) and V_(L)together forms a single antigen-binding site. For the structure andproperties of the different classes of antibodies, see, e.g., Basic andClinical Immunology, 8th edition, Daniel P. Stites, Abba I. Terr andTristram G. Parslow (eds.), Appleton & Lange, Norwalk, Conn., 1994, page71, and Chapter 6.

The L chain from any vertebrate species can be assigned to one of twoclearly distinct types, called kappa (κ) and lambda (λ), based on theamino acid sequences of their constant domains (C_(L)). Depending on theamino acid sequence of the constant domain of their heavy chains(C_(H)), immunoglobulins can be assigned to different classes orisotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG,and IgM, having heavy chains designated alpha (α), delta (δ)□, epsilon(ε), gamma (γ□) and mu (μ)□, respectively. The γ and α classes arefurther divided into subclasses on the basis of relatively minordifferences in C_(H) sequence and function, e.g., humans express thefollowing subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

The term “variable” refers to the fact that certain segments of the Vdomains differ extensively in sequence among antibodies. The V domainmediates antigen binding and defines specificity of a particularantibody for its particular antigen. However, the variability is notevenly distributed across the 110-amino acid span of the variableregions. Instead, the V regions consist of relatively invariantstretches called framework regions (FRs) of 15-30 amino acids separatedby shorter regions of extreme variability called “hypervariable regions”that are each 9-12 amino acids long. The variable regions of nativeheavy and light chains each comprise four FRs, largely adopting aβ-sheet configuration, connected by three hypervariable regions, whichform loops connecting, and in some cases forming part of, the □β-sheetstructure. The hypervariable regions in each chain are held together inclose proximity by the FRs and, with the hypervariable regions from theother chain, contribute to the formation of the antigen-binding site ofantibodies (see Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991)). The constant domains are not involved directly inbinding an antibody to an antigen, but exhibit various effectorfunctions, such as participation of the antibody in antibody dependentcellular cytotoxicity (ADCC).

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody that are responsible for antigen binding.The hypervariable region generally comprises amino acid residues from a“complementarity determining region” or “CDR” (e.g., around aboutresidues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the V_(L), and aroundabout 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the V_(H) when numberedin accordance with the Kabat numbering system; Kabat et al., Sequencesof Proteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)); and/or thoseresidues from a “hypervariable loop” (e.g., residues 24-34 (L1), 50-56(L2) and 89-97 (L3) in the V_(L), and 26-32 (H1), 52-56 (H2) and 95-101(H3) in the V_(H) when numbered in accordance with the Chothia numberingsystem; Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); and/orthose residues from a “hypervariable loop”/CDR (e.g., residues 27-38(L1), 56-65 (L2) and 105-120 (L3) in the V_(L), and 27-38 (H1), 56-65(H2) and 105-120 (H3) in the V_(L) when numbered in accordance with theIMGT numbering system; Lefranc, M. P. et al. Nucl. Acids Res. 27:209-212(1999), Ruiz, M. e al. Nucl. Acids Res. 28:219-221 (2000)). Optionallythe antibody has symmetrical insertions at one or more of the followingpoints 28, 36 (L1), 63, 74-75 (L2) and 123 (L3) in the V_(L), and 28, 36(H1), 63, 74-75 (H2) and 123 (H3) in the V_(H) when numbered inaccordance with AHo; Honneger, A. and Plunkthun, A. J. Mol. Biol.309:657-670 (2001)).

By “germline nucleic acid residue” is meant the nucleic acid residuethat naturally occurs in a germline gene encoding a constant or variableregion. “Germline gene” is the DNA found in a germ cell (i.e., a celldestined to become an egg or in the sperm). A “germline mutation” refersto a heritable change in a particular DNA that has occurred in a germcell or the zygote at the single-cell stage, and when transmitted tooffspring, such a mutation is incorporated in every cell of the body. Agermline mutation is in contrast to a somatic mutation which is acquiredin a single body cell. In some cases, nucleotides in a germline DNAsequence encoding for a variable region are mutated (i.e., a somaticmutation) and replaced with a different nucleotide.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations that include different antibodies directed againstdifferent determinants (epitopes), each monoclonal antibody is directedagainst a single determinant on the antigen. In addition to theirspecificity, the monoclonal antibodies are advantageous in that they maybe synthesized uncontaminated by other antibodies. The modifier“monoclonal” is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies useful in the present invention may be prepared by thehybridoma methodology first described by Kohler et al., Nature, 256:495(1975), or may be made using recombinant DNA methods in bacterial,eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567).The “monoclonal antibodies” may also be isolated from phage antibodylibraries using the techniques described in Clackson et al., Nature,352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991),for example.

In some aspects, the alternative EBV immortalization method described inWO2004/076677 is used. Using this method, B-cells producing the antibodyof the invention can be transformed with EBV in the presence of apolyclonal B cell activator. Transformation with EBV is a standardtechnique and can easily be adapted to include polyclonal B cellactivators. Additional stimulants of cellular growth and differentiationmay be added during the transformation step to further enhance theefficiency. These stimulants may be cytokines such as IL-2 and IL-15. Ina particularly preferred aspect, IL-2 is added during theimmortalization step to further improve the efficiency ofimmortalization, but its use is not essential.

The monoclonal antibodies herein include “chimeric” antibodies in whicha portion of the heavy and/or light chain is identical with orhomologous to corresponding sequences in antibodies derived from aparticular species or belonging to a particular antibody class orsubclass, while the remainder of the chain(s) is identical with orhomologous to corresponding sequences in antibodies derived from anotherspecies or belonging to another antibody class or subclass, as well asfragments of such antibodies, so long as they exhibit the desiredbiological activity (see U.S. Pat. No. 4,816,567; and Morrison et al.,Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). The present inventionprovides variable region antigen-binding sequences derived from humanantibodies. Accordingly, chimeric antibodies of primary interest hereininclude antibodies having one or more human antigen binding sequences(e.g., CDRs) and containing one or more sequences derived from anon-human antibody, e.g., an FR or C region sequence. In addition,chimeric antibodies of primary interest herein include those comprisinga human variable region antigen binding sequence of one antibody classor subclass and another sequence, e.g., FR or C region sequence, derivedfrom another antibody class or subclass. Chimeric antibodies of interestherein also include those containing variable region antigen-bindingsequences related to those described herein or derived from a differentspecies, such as a non-human primate (e.g., Old World Monkey, Ape, etc).Chimeric antibodies also include primatized and humanized antibodies.

Furthermore, chimeric antibodies may comprise residues that are notfound in the recipient antibody or in the donor antibody. Thesemodifications are made to further refine antibody performance. Forfurther details, see Jones et al., Nature 321:522-525 (1986); Riechmannet al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.2:593-596 (1992).

A “humanized antibody” is generally considered to be a human antibodythat has one or more amino acid residues introduced into it from asource that is non-human. These non-human amino acid residues are oftenreferred to as “import” residues, which are typically taken from an“import” variable region. Humanization is traditionally performedfollowing the method of Winter and co-workers (Jones et al., Nature,321:522-525 (1986); Reichmann et al., Nature, 332:323-327 (1988);Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting importhypervariable region sequences for the corresponding sequences of ahuman antibody. Accordingly, such “humanized” antibodies are chimericantibodies (U.S. Pat. No. 4,816,567) wherein substantially less than anintact human variable region has been substituted by the correspondingsequence from a non-human species.

A “human antibody” is an antibody containing only sequences present inan antibody naturally produced by a human. However, as used herein,human antibodies may comprise residues or modifications not found in anaturally occurring human antibody, including those modifications andvariant sequences described herein. These are typically made to furtherrefine or enhance antibody performance.

An “intact” antibody is one that comprises an antigen-binding site aswell as a C_(L) and at least heavy chain constant domains, C_(H) 1,C_(H) 2 and C_(H) 3. The constant domains may be native sequenceconstant domains (e.g., human native sequence constant domains) or aminoacid sequence variant thereof. Preferably, the intact antibody has oneor more effector functions.

An “antibody fragment” comprises a portion of an intact antibody,preferably the antigen binding or variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂, andFv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870;Zapata et al., Protein Eng. 8(10): 1057-1062 [1995]), single-chainantibody molecules; and multispecific antibodies formed from antibodyfragments.

The phrase “functional fragment or analog” of an antibody is a compoundhaving qualitative biological activity in common with a full-lengthantibody. For example, a functional fragment or analog of an anti-IgEantibody is one that can bind to an IgE immunoglobulin in such a mannerso as to prevent or substantially reduce the ability of such moleculefrom having the ability to bind to the high affinity receptor, Fc_(ε)RI.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, and a residual “Fc” fragment, adesignation reflecting the ability to crystallize readily. The Fabfragment consists of an entire L chain along with the variable regiondomain of the H chain (V_(H)), and the first constant domain of oneheavy chain (C_(H) 1). Each Fab fragment is monovalent with respect toantigen binding, i.e., it has a single antigen-binding site. Pepsintreatment of an antibody yields a single large F(ab′)₂ fragment thatroughly corresponds to two disulfide linked Fab fragments havingdivalent antigen-binding activity and is still capable of cross-linkingantigen. Fab′ fragments differ from Fab fragments by having additionalfew residues at the carboxy terminus of the C_(H)1 domain including oneor more cysteines from the antibody hinge region. Fab′-SH is thedesignation herein for Fab′ in which the cysteine residue(s) of theconstant domains bear a free thiol group. F(ab′)₂ antibody fragmentsoriginally were produced as pairs of Fab′ fragments that have hingecysteines between them. Other chemical couplings of antibody fragmentsare also known.

The “Fc” fragment comprises the carboxy-terminal portions of both Hchains held together by disulfides. The effector functions of antibodiesare determined by sequences in the Fc region, which region is also thepart recognized by Fc receptors (FcR) found on certain types of cells.

“Fv” is the minimum antibody fragment that contains a completeantigen-recognition and -binding site. This fragment consists of a dimerof one heavy- and one light-chain variable region domain in tight,non-covalent association. From the folding of these two domains emanatesix hypervariable loops (three loops each from the H and L chain) thatcontribute the amino acid residues for antigen binding and conferantigen binding specificity to the antibody. However, even a singlevariable region (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

“Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibodyfragments that comprise the V_(H) and V_(L) antibody domains connectedinto a single polypeptide chain. Preferably, the sFv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains thatenables the sFv to form the desired structure for antigen binding. For areview of sFv, see Pluckthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994); Borrebaeck 1995, infra.

The term “diabodies” refers to small antibody fragments prepared byconstructing sFv fragments (see preceding paragraph) with short linkers(about 5-10 residues) between the V_(H) and V_(L) domains such thatinter-chain but not intra-chain pairing of the V domains is achieved,resulting in a bivalent fragment, i.e., fragment having twoantigen-binding sites. Bispecific diabodies are heterodimers of two“crossover” sFv fragments in which the V_(H) and V_(L) domains of thetwo antibodies are present on different polypeptide chains. Diabodiesare described more fully in, for example, EP 404,097; WO 93/11161; andHollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

Domain antibodies (dAbs), which can be produced in fully human form, arethe smallest known antigen-binding fragments of antibodies, ranging from11 kDa to 15 kDa. dAbs are the robust variable regions of the heavy andlight chains of immunoglobulins (VH and VL respectively). They arehighly expressed in microbial cell culture, show favourable biophysicalproperties including solubility and temperature stability, and are wellsuited to selection and affinity maturation by in vitro selectionsystems such as phage display. dAbs are bioactive as monomers and, owingto their small size and inherent stability, can be formatted into largermolecules to create drugs with prolonged serum half-lives or otherpharmacological activities. Examples of this technology have beendescribed in WO9425591 for antibodies derived from Camelidae heavy chainIg, as well in US20030130496 describing the isolation of single domainfully human antibodies from phage libraries.

As used herein, an antibody that “internalizes” is one that is taken upby (i.e., enters) the cell upon binding to an antigen on a mammaliancell (e.g., a cell surface polypeptide or receptor). The internalizingantibody will of course include antibody fragments, human or chimericantibody, and antibody conjugates. For certain therapeutic applications,internalization in vivo is contemplated. The number of antibodymolecules internalized will be sufficient or adequate to kill a cell orinhibit its growth, especially an infected cell. Depending on thepotency of the antibody or antibody conjugate, in some instances, theuptake of a single antibody molecule into the cell is sufficient to killthe target cell to which the antibody binds. For example, certain toxinsare highly potent in killing such that internalization of one moleculeof the toxin conjugated to the antibody is sufficient to kill theinfected cell.

As used herein, an antibody is said to be “immunospecific,” “specificfor” or to “specifically bind” an antigen if it reacts at a detectablelevel with the antigen, preferably with an affinity constant, K_(a), ofgreater than or equal to about 10⁴ M⁻¹, or greater than or equal toabout 10⁵ M⁻¹, greater than or equal to about 10⁶ M⁻¹, greater than orequal to about 10⁷ M⁻¹, or greater than or equal to 10⁸ M⁻¹. Affinity ofan antibody for its cognate antigen is also commonly expressed as adissociation constant K_(D), and in certain embodiments, HIV antibodyspecifically binds to an HIV1 polypeptide if it binds with a K_(D) ofless than or equal to 10⁻⁴ M, less than or equal to about 10⁻⁵ M, lessthan or equal to about 10⁻⁶ M, less than or equal to 10⁻⁷ M, or lessthan or equal to 10⁻⁸ M. Affinities of antibodies can be readilydetermined using conventional techniques, for example, those describedby Scatchard et al. (Ann. N. Y. Acad. Sci. USA 51:660 (1949)).

Binding properties of an antibody to antigens, cells or tissues thereofmay generally be determined and assessed using immunodetection methodsincluding, for example, immunofluorescence-based assays, such asimmuno-histochemistry (IHC) and/or fluorescence-activated cell sorting(FACS).

An antibody having a “biological characteristic” of a designatedantibody is one that possesses one or more of the biologicalcharacteristics of that antibody which distinguish it from otherantibodies. For example, in certain embodiments, an antibody with abiological characteristic of a designated antibody will bind the sameepitope as that bound by the designated antibody and/or have a commoneffector function as the designated antibody.

The term “antagonist” antibody is used in the broadest sense, andincludes an antibody that partially or fully blocks, inhibits, orneutralizes a biological activity of an epitope, polypeptide, or cellthat it specifically binds. Methods for identifying antagonistantibodies may comprise contacting a polypeptide or cell specificallybound by a candidate antagonist antibody with the candidate antagonistantibody and measuring a detectable change in one or more biologicalactivities normally associated with the polypeptide or cell.

An “antibody that inhibits the growth of infected cells” or a “growthinhibitory” antibody is one that binds to and results in measurablegrowth inhibition of infected cells expressing or capable of expressingan HIV1 epitope bound by an antibody. Preferred growth inhibitoryantibodies inhibit growth of infected cells by greater than 20%,preferably from about 20% to about 50%, and even more preferably, bygreater than 50% (e.g., from about 50% to about 100%) as compared to theappropriate control, the control typically being infected cells nottreated with the antibody being tested. Growth inhibition can bemeasured at an antibody concentration of about 0.1 to 30 μg/ml or about0.5 nM to 200 nM in cell culture, where the growth inhibition isdetermined 1-10 days after exposure of the infected cells to theantibody. Growth inhibition of infected cells in vivo can be determinedin various ways known in the art.

The antibody is growth inhibitory in vivo if administration of theantibody at about 1 μg/kg to about 100 mg/kg body weight results inreduction the percent of infected cells or total number of infectedcells within about 5 days to 3 months from the first administration ofthe antibody, preferably within about 5 to 30 days.

An antibody that “induces apoptosis” is one which induces programmedcell death as determined by binding of annexin V, fragmentation of DNA,cell shrinkage, dilation of endoplasmic reticulum, cell fragmentation,and/or formation of membrane vesicles (called apoptotic bodies).Preferably the cell is an infected cell. Various methods are availablefor evaluating the cellular events associated with apoptosis. Forexample, phosphatidyl serine (PS) translocation can be measured byannexin binding; DNA fragmentation can be evaluated through DNAladdering; and nuclear/chromatin condensation along with DNAfragmentation can be evaluated by any increase in hypodiploid cells.Preferably, the antibody that induces apoptosis is one that results inabout 2 to 50 fold, preferably about 5 to 50 fold, and most preferablyabout 10 to 50 fold, induction of annexin binding relative to untreatedcell in an annexin binding assay.

Antibody “effector functions” refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody, and vary with the antibodyisotype. Examples of antibody effector functions include: C1q bindingand complement dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor); and B cellactivation.

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to aform of cytotoxicity in which secreted Ig bound to Fc receptors (FcRs)present on certain cytotoxic cells (e.g., Natural Killer (NK) cells,neutrophils, and macrophages) enable these cytotoxic effector cells tobind specifically to an antigen-bearing target cell and subsequentlykill the target cell with cytotoxins. The antibodies “arm” the cytotoxiccells and are required for such killing. The primary cells for mediatingADCC, NK cells, express FcγRIII only, whereas monocytes express FcγRI,FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarizedin Table 4 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92(1991). To assess ADCC activity of a molecule of interest, an in vitroADCC assay, such as that described in U.S. Pat. No. 5,500,362 or U.S.Pat. No. 5,821,337 may be performed. Useful effector cells for suchassays include peripheral blood mononuclear cells (PBMC) and NaturalKiller (NK) cells.

Alternatively, or additionally, ADCC activity of the molecule ofinterest may be assessed in vivo, e.g., in a animal model such as thatdisclosed in Clynes et al., Proc. Natl. Acad. Sci. (USA) 95:652-656(1998).

“Fc receptor” or “FcR” describes a receptor that binds to the Fc regionof an antibody. In certain embodiments, the FcR is a native sequencehuman FcR. Moreover, a preferred FcR is one that binds an IgG antibody(a gamma receptor) and includes receptors of the FcγRI, FcγRII, andFcγRIII subclasses, including allelic variants and alternatively splicedforms of these receptors. FCγRII receptors include FcγRIIA (an“activating receptor”) and FcγRIIB (an “inhibiting receptor”), whichhave similar amino acid sequences that differ primarily in thecytoplasmic domains thereof. Activating receptor FcγRIIA contains animmunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmicdomain. Inhibiting receptor FcγRIIB contains an immunoreceptortyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (seereview M. in Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs arereviewed in Ravetch and Kinet. Annu. Rev. Immunol 9:457-92 (1991); Capelet al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin.Med. 126:330-41 (1995). Other FcRs, including those to be identified inthe future, are encompassed by the term “FcR” herein. The term alsoincludes the neonatal receptor, FcRn, which is responsible for thetransfer of maternal IgGs to the fetus (Guyer et al., J. Immunol.117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)).

“Human effector cells” are leukocytes that express one or more FcRs andperform effector functions. Preferably, the cells express at leastFcγRIII and perform ADCC effector function. Examples of human leukocytesthat mediate ADCC include PBMC, NK cells, monocytes, cytotoxic T cellsand neutrophils; with PBMCs and NK cells being preferred. The effectorcells may be isolated from a native source, e.g., from blood.

“Complement dependent cytotoxicity” or “CDC” refers to the lysis of atarget cell in the presence of complement. Activation of the classicalcomplement pathway is initiated by the binding of the first component ofthe complement system (C1q) to antibodies (of the appropriate subclass)that are bound to their cognate antigen. To assess complementactivation, a CDC assay, e.g., as described in Gazzano-Santoro et al.,J. Immunol. Methods 202:163 (1996), may be performed.

A “mammal” for purposes of treating an infection, refers to any mammal,including humans, domestic and farm animals, and zoo, sports, or petanimals, such as dogs, cats, cattle, horses, sheep, pigs, goats,rabbits, etc. Preferably, the mammal is human.

“Treating” or “treatment” or “alleviation” refers to both therapeutictreatment and prophylactic or preventative measures; wherein the objectis to prevent or slow down (lessen) the targeted pathologic condition ordisorder. Those in need of treatment include those already with thedisorder as well as those prone to have the disorder or those in whomthe disorder is to be prevented. A subject or mammal is successfully“treated” for an infection if, after receiving a therapeutic amount ofan antibody according to the methods of the present invention, thepatient shows observable and/or measurable reduction in or absence ofone or more of the following: reduction in the number of infected cellsor absence of the infected cells; reduction in the percent of totalcells that are infected; and/or relief to some extent, one or more ofthe symptoms associated with the specific infection; reduced morbidityand mortality, and improvement in quality of life issues. The aboveparameters for assessing successful treatment and improvement in thedisease are readily measurable by routine procedures familiar to aphysician.

The term “therapeutically effective amount” refers to an amount of anantibody or a drug effective to “treat” a disease or disorder in asubject or mammal. See preceding definition of “treating.”

“Chronic” administration refers to administration of the agent(s) in acontinuous mode as opposed to an acute mode, so as to maintain theinitial therapeutic effect (activity) for an extended period of time.“Intermittent” administration is treatment that is not consecutivelydone without interruption, but rather is cyclic in nature.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers that are nontoxic to the cell or mammal beingexposed thereto at the dosages and concentrations employed. Often thephysiologically acceptable carrier is an aqueous pH buffered solution.Examples of physiologically acceptable carriers include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid; low molecular weight (less than about 10 residues)polypeptide; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, arginine or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugaralcohols such as mannitol or sorbitol; salt-forming counterions such assodium; and/or nonionic surfactants such as TWEEN™ polyethylene glycol(PEG), and PLURONICS™.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g.,At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³² and radioactiveisotopes of Lu), chemotherapeutic agents e.g., methotrexate, adriamicin,vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin,melphalan, mitomycin C, chlorambucil, daunorubicin or otherintercalating agents, enzymes and fragments thereof such as nucleolyticenzymes, antibiotics, and toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof, and the variousantitumor or anticancer agents disclosed below. Other cytotoxic agentsare described below.

A “growth inhibitory agent” when used herein refers to a compound orcomposition which inhibits growth of a cell, either in vitro or in vivo.Examples of growth inhibitory agents include agents that block cellcycle progression, such as agents that induce G1 arrest and M-phasearrest. Classical M-phase blockers include the vinca alkaloids(vincristine, vinorelbine and vinblastine), taxanes, and topoisomeraseII inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide,and bleomycin. Those agents that arrest G1 also spill over into S-phasearrest, for example, DNA alkylating agents such as tamoxifen,prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate,5-fluorouracil, and ara-C. Further information can be found in TheMolecular Basis of Cancer, Mendelsohn and Israel, eds., Chapter 1,entitled “Cell cycle regulation, oncogenes, and antineoplastic drugs” byMurakami et al. (W B Saunders: Philadelphia, 1995), especially p. 13.The taxanes (paclitaxel and docetaxel) are anticancer drugs both derivedfrom the yew tree. Docetaxel (TAXOTERE™, Rhone-Poulenc Rorer), derivedfrom the European yew, is a semisynthetic analogue of paclitaxel(TAXOL®, Bristol-Myers Squibb). Paclitaxel and docetaxel promote theassembly of microtubules from tubulin dimers and stabilize microtubulesby preventing depolymerization, which results in the inhibition ofmitosis in cells.

“Label” as used herein refers to a detectable compound or compositionthat is conjugated directly or indirectly to the antibody so as togenerate a “labeled” antibody. The label may be detectable by itself(e.g., radioisotope labels or fluorescent labels) or, in the case of anenzymatic label, may catalyze chemical alteration of a substratecompound or composition that is detectable.

The term “epitope tagged” as used herein refers to a chimericpolypeptide comprising a polypeptide fused to a “tag polypeptide.” Thetag polypeptide has enough residues to provide an epitope against whichan antibody can be made, yet is short enough such that it does notinterfere with activity of the polypeptide to which it is fused. The tagpolypeptide is also preferably fairly unique so that the antibody doesnot substantially cross-react with other epitopes. Suitable tagpolypeptides generally have at least six amino acid residues and usuallybetween about 8 and 50 amino acid residues (preferably, between about 10and 20 amino acid residues).

A “small molecule” is defined herein to have a molecular weight belowabout 500 Daltons.

The terms “nucleic acid” and “polynucleotide” are used interchangeablyherein to refer to single- or double-stranded RNA, DNA, or mixedpolymers. Polynucleotides may include genomic sequences, extra-genomicand plasmid sequences, and smaller engineered gene segments thatexpress, or may be adapted to express polypeptides.

An “isolated nucleic acid” is a nucleic acid that is substantiallyseparated from other genome DNA sequences as well as proteins orcomplexes such as ribosomes and polymerases, which naturally accompany anative sequence. The term embraces a nucleic acid sequence that has beenremoved from its naturally occurring environment, and includesrecombinant or cloned DNA isolates and chemically synthesized analoguesor analogues biologically synthesized by heterologous systems. Asubstantially pure nucleic acid includes isolated forms of the nucleicacid. Of course, this refers to the nucleic acid as originally isolatedand does not exclude genes or sequences later added to the isolatednucleic acid by the hand of man.

The term “polypeptide” is used in its conventional meaning, i.e., as asequence of amino acids. The polypeptides are not limited to a specificlength of the product. Peptides, oligopeptides, and proteins areincluded within the definition of polypeptide, and such terms may beused interchangeably herein unless specifically indicated otherwise.This term also does not refer to or exclude post-expressionmodifications of the polypeptide, for example, glycosylations,acetylations, phosphorylations and the like, as well as othermodifications known in the art, both naturally occurring andnon-naturally occurring. A polypeptide may be an entire protein, or asubsequence thereof. Particular polypeptides of interest in the contextof this invention are amino acid subsequences comprising CDRs and beingcapable of binding an antigen or HIV-infected cell.

An “isolated polypeptide” is one that has been identified and separatedand/or recovered from a component of its natural environment. Inpreferred embodiments, the isolated polypeptide will be purified (1) togreater than 95% by weight of polypeptide as determined by the Lowrymethod, and most preferably more than 99% by weight, (2) to a degreesufficient to obtain at least 15 residues of N-terminal or internalamino acid sequence by use of a spinning cup sequenator, or (3) tohomogeneity by SDS-PAGE under reducing or non-reducing conditions usingCoomassie blue or, preferably, silver stain. Isolated polypeptideincludes the polypeptide in situ within recombinant cells since at leastone component of the polypeptide's natural environment will not bepresent. Ordinarily, however, isolated polypeptide will be prepared byat least one purification step.

A “native sequence” polynucleotide is one that has the same nucleotidesequence as a polynucleotide derived from nature. A “native sequence”polypeptide is one that has the same amino acid sequence as apolypeptide (e.g., antibody) derived from nature (e.g., from anyspecies). Such native sequence polynucleotides and polypeptides can beisolated from nature or can be produced by recombinant or syntheticmeans.

A polynucleotide “variant,” as the term is used herein, is apolynucleotide that typically differs from a polynucleotide specificallydisclosed herein in one or more substitutions, deletions, additionsand/or insertions. Such variants may be naturally occurring or may besynthetically generated, for example, by modifying one or more of thepolynucleotide sequences of the invention and evaluating one or morebiological activities of the encoded polypeptide as described hereinand/or using any of a number of techniques well known in the art.

A polypeptide “variant,” as the term is used herein, is a polypeptidethat typically differs from a polypeptide specifically disclosed hereinin one or more substitutions, deletions, additions and/or insertions.Such variants may be naturally occurring or may be syntheticallygenerated, for example, by modifying one or more of the abovepolypeptide sequences of the invention and evaluating one or morebiological activities of the polypeptide as described herein and/orusing any of a number of techniques well known in the art.

Modifications may be made in the structure of the polynucleotides andpolypeptides of the present invention and still obtain a functionalmolecule that encodes a variant or derivative polypeptide with desirablecharacteristics. When it is desired to alter the amino acid sequence ofa polypeptide to create an equivalent, or even an improved, variant orportion of a polypeptide of the invention, one skilled in the art willtypically change one or more of the codons of the encoding DNA sequence.

For example, certain amino acids may be substituted for other aminoacids in a protein structure without appreciable loss of its ability tobind other polypeptides (e.g., antigens) or cells. Since it is thebinding capacity and nature of a protein that defines that protein'sbiological functional activity, certain amino acid sequencesubstitutions can be made in a protein sequence, and, of course, it'sunderlying DNA coding sequence, and nevertheless obtain a protein withlike properties. It is thus contemplated that various changes may bemade in the peptide sequences of the disclosed compositions, orcorresponding DNA sequences that encode said peptides withoutappreciable loss of their biological utility or activity.

In many instances, a polypeptide variant will contain one or moreconservative substitutions. A “conservative substitution” is one inwhich an amino acid is substituted for another amino acid that hassimilar properties, such that one skilled in the art of peptidechemistry would expect the secondary structure and hydropathic nature ofthe polypeptide to be substantially unchanged.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982). It is accepted thatthe relative hydropathic character of the amino acid contributes to thesecondary structure of the resultant protein, which in turn defines theinteraction of the protein with other molecules, for example, enzymes,substrates, receptors, DNA, antibodies, antigens, and the like. Eachamino acid has been assigned a hydropathic index on the basis of itshydrophobicity and charge characteristics (Kyte and Doolittle, 1982).These values are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9);alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e. still obtaina biological functionally equivalent protein. In making such changes,the substitution of amino acids whose hydropathic indices are within ±2is preferred, those within ±1 are particularly preferred, and thosewithin ±0.5 are even more particularly preferred. It is also understoodin the art that the substitution of like amino acids can be madeeffectively on the basis of hydrophilicity. U.S. Pat. No. 4,554,101states that the greatest local average hydrophilicity of a protein, asgoverned by the hydrophilicity of its adjacent amino acids, correlateswith a biological property of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). It isunderstood that an amino acid can be substituted for another having asimilar hydrophilicity value and still obtain a biologically equivalent,and in particular, an immunologically equivalent protein. In suchchanges, the substitution of amino acids whose hydrophilicity values arewithin ±2 is preferred, those within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally thereforebased on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions that take various of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine.

Amino acid substitutions may further be made on the basis of similarityin polarity, charge, solubility, hydrophobicity, hydrophilicity and/orthe amphipathic nature of the residues. For example, negatively chargedamino acids include aspartic acid and glutamic acid; positively chargedamino acids include lysine and arginine; and amino acids with unchargedpolar head groups having similar hydrophilicity values include leucine,isoleucine and valine; glycine and alanine; asparagine and glutamine;and serine, threonine, phenylalanine and tyrosine. Other groups of aminoacids that may represent conservative changes include: (1) ala, pro,gly, glu, asp, gin, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile,leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. Avariant may also, or alternatively, contain nonconservative changes. Ina preferred embodiment, variant polypeptides differ from a nativesequence by substitution, deletion or addition of five amino acids orfewer. Variants may also (or alternatively) be modified by, for example,the deletion or addition of amino acids that have minimal influence onthe immunogenicity, secondary structure and hydropathic nature of thepolypeptide.

Polypeptides may comprise a signal (or leader) sequence at theN-terminal end of the protein, which co-translationally orpost-translationally directs transfer of the protein. The polypeptidemay also be conjugated to a linker or other sequence for ease ofsynthesis, purification or identification of the polypeptide (e.g.,poly-His), or to enhance binding of the polypeptide to a solid support.For example, a polypeptide may be conjugated to an immunoglobulin Fcregion.

When comparing polynucleotide and polypeptide sequences, two sequencesare said to be “identical” if the sequence of nucleotides or amino acidsin the two sequences is the same when aligned for maximumcorrespondence, as described below. Comparisons between two sequencesare typically performed by comparing the sequences over a comparisonwindow to identify and compare local regions of sequence similarity. A“comparison window” as used herein, refers to a segment of at leastabout 20 contiguous positions, usually 30 to about 75, 40 to about 50,in which a sequence may be compared to a reference sequence of the samenumber of contiguous positions after the two sequences are optimallyaligned.

Optimal alignment of sequences for comparison may be conducted using theMegalign program in the Lasergene suite of bioinformatics software(DNASTAR, Inc., Madison, Wis.), using default parameters. This programembodies several alignment schemes described in the followingreferences: Dayhoff, M. O. (1978) A model of evolutionary change inproteins—Matrices for detecting distant relationships. In Dayhoff, M. O.(ed.) Atlas of Protein Sequence and Structure, National BiomedicalResearch Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; HeinJ. (1990) Unified Approach to Alignment and Phylogenes pp. 626-645Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.;Higgins, D. G. and Sharp, P. M. (1989) CABIOS 5:151-153; Myers, E. W.and Muller W. (1988) CABIOS 4:11-17; Robinson, E. D. (1971) Comb. Theor11:105; Santou, N. Nes, M. (1987) Mol. Biol. Evol. 4:406-425; Sneath, P.H. A. and Sokal, R. R. (1973) Numerical Taxonomy—the Principles andPractice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.;Wilbur, W. J. and Lipman, D. J. (1983) Proc. Natl. Acad., Sci. USA80:726-730.

Alternatively, optimal alignment of sequences for comparison may beconducted by the local identity algorithm of Smith and Waterman (1981)Add. APL. Math 2:482, by the identity alignment algorithm of Needlemanand Wunsch (1970) J. Mol. Biol. 48:443, by the search for similaritymethods of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT,BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package,Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.), or byinspection.

One preferred example of algorithms that are suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al. (1977)Nucl. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol.215:403-410, respectively. BLAST and BLAST 2.0 can be used, for examplewith the parameters described herein, to determine percent sequenceidentity for the polynucleotides and polypeptides of the invention.Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information.

In one illustrative example, cumulative scores can be calculated using,for nucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). Extension of the word hits in each direction arehalted when: the cumulative alignment score falls off by the quantity Xfrom its maximum achieved value; the cumulative score goes to zero orbelow, due to the accumulation of one or more negative-scoring residuealignments; or the end of either sequence is reached. The BLASTalgorithm parameters W, T and X determine the sensitivity and speed ofthe alignment. The BLASTN program (for nucleotide sequences) uses asdefaults a wordlength (W) of 11, and expectation (E) of 10, and theBLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl.Acad. Sci. USA 89:10915) alignments, (B) of 50, expectation (E) of 10,M=5, N=−4 and a comparison of both strands.

For amino acid sequences, a scoring matrix can be used to calculate thecumulative score. Extension of the word hits in each direction arehalted when: the cumulative alignment score falls off by the quantity Xfrom its maximum achieved value; the cumulative score goes to zero orbelow, due to the accumulation of one or more negative-scoring residuealignments; or the end of either sequence is reached. The BLASTalgorithm parameters W, T and X determine the sensitivity and speed ofthe alignment.

In one approach, the “percentage of sequence identity” is determined bycomparing two optimally aligned sequences over a window of comparison ofat least 20 positions, wherein the portion of the polynucleotide orpolypeptide sequence in the comparison window may comprise additions ordeletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent,or 10 to 12 percent, as compared to the reference sequences (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical nucleic acid bases or amino acidresidues occur in both sequences to yield the number of matchedpositions, dividing the number of matched positions by the total numberof positions in the reference sequence (i.e., the window size) andmultiplying the results by 100 to yield the percentage of sequenceidentity.

“Homology” refers to the percentage of residues in the polynucleotide orpolypeptide sequence variant that are identical to the non-variantsequence after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent homology. In particularembodiments, polynucleotide and polypeptide variants have at least 70%,at least 75%, at least 80%, at least 900%, at least 95%, at least 98%,or at least 99% polynucleotide or polypeptide homology with apolynucleotide or polypeptide described herein.

“Vector” includes shuttle and expression vectors. Typically, the plasmidconstruct will also include an origin of replication (e.g., the Co1E1origin of replication) and a selectable marker (e.g., ampicillin ortetracycline resistance), for replication and selection, respectively,of the plasmids in bacteria. An “expression vector” refers to a vectorthat contains the necessary control sequences or regulatory elements forexpression of the antibodies including antibody fragment of theinvention, in bacterial or eukaryotic cells. Suitable vectors aredisclosed below. As used in this specification and the appended claims,the singular forms “a,” “an” and “the” include plural references unlessthe content clearly dictates otherwise.

The invention also includes nucleic acid sequences encoding part or allof the light and heavy chains and CDRs of the present invention. Due toredundancy of the genetic code, variants of these sequences will existthat encode the same amino acid sequences.

Variant antibodies are also included within the scope of the invention.Thus, variants of the sequences recited in the application are alsoincluded within the scope of the invention. Further variants of theantibody sequences having improved affinity may be obtained usingmethods known in the art and are included within the scope of theinvention. For example, amino acid substitutions may be used to obtainantibodies with further improved affinity. Alternatively, codonoptimization of the nucleotide sequence may be used to improve theefficiency of translation in expression systems for the production ofthe antibody.

Preferably, such variant antibody sequences will share 70% or more (i.e.80, 85, 90, 95, 97, 98, 99% or more) sequence identity with thesequences recited in the application. Preferably such sequence identityis calculated with regard to the full length of the reference sequence(i.e. the sequence recited in the application). Preferably, percentageidentity, as referred to herein, is as determined using BLAST version2.1.3 using the default parameters specified by the NCBI (the NationalCenter for Biotechnology Information; http://www.ncbi.nlm.nih.gov/)[Blosum 62 matrix; gap open penalty=11 and gap extension penalty=1].

Further included within the scope of the invention are vectors such asexpression vectors, comprising a nucleic acid sequence according to theinvention. Cells transformed with such vectors are also included withinthe scope of the invention.

As will be understood by the skilled artisan, general description ofantibodies herein and methods of preparing and using the same also applyto individual antibody polypeptide constituents and antibody fragments.

The antibodies of the present invention may be polyclonal or monoclonalantibodies. However, in preferred embodiments, they are monoclonal. Inparticular embodiments, antibodies of the present invention are humanantibodies. Methods of producing polyclonal and monoclonal antibodiesare known in the art and described generally, e.g., in U.S. Pat. No.6,824,780.

Typically, the antibodies of the present invention are producedrecombinantly, using vectors and methods available in the art, asdescribed further below. Human antibodies may also be generated by invitro activated B cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275).

Human antibodies may also be produced in transgenic animals (e.g., mice)that are capable of producing a full repertoire of human antibodies inthe absence of endogenous immunoglobulin production. For example, it hasbeen described that the homozygous deletion of the antibody heavy-chainjoining region (J_(H)) gene in chimeric and germ-line mutant miceresults in complete inhibition of endogenous antibody production.Transfer of the human germ-line immunoglobulin gene array into suchgerm-line mutant mice results in the production of human antibodies uponantigen challenge. See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci.USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993);Bruggemann et al., Year in Immuno., 7:33 (1993); U.S. Pat. Nos.5,545,806, 5,569,825, 5,591,669 (all of GenPharm); U.S. Pat. No.5,545,807; and WO 97/17852. Such animals may be genetically engineeredto produce human antibodies comprising a polypeptide of the presentinvention.

In certain embodiments, antibodies of the present invention are chimericantibodies that comprise sequences derived from both human and non-humansources. In particular embodiments, these chimeric antibodies arehumanized or Primatized™. In practice, humanized antibodies aretypically human antibodies in which some hypervariable region residuesand possibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

In the context of the present invention, chimeric antibodies alsoinclude human antibodies wherein the human hypervariable region or oneor more CDRs are retained, but one or more other regions of sequencehave been replaced by corresponding sequences from a non-human animal.

The choice of non-human sequences, both light and heavy, to be used inmaking the chimeric antibodies is important to reduce antigenicity andhuman anti-non-human antibody responses when the antibody is intendedfor human therapeutic use. It is further important that chimericantibodies retain high binding affinity for the antigen and otherfavorable biological properties. To achieve this goal, according to apreferred method, chimeric antibodies are prepared by a process ofanalysis of the parental sequences and various conceptual chimericproducts using three-dimensional models of the parental human andnon-human sequences. Three-dimensional immunoglobulin models arecommonly available and are familiar to those skilled in the art.Computer programs are available which illustrate and display probablethree-dimensional conformational structures of selected candidateimmunoglobulin sequences.

Inspection of these displays permits analysis of the likely role of theresidues in the functioning of the candidate immunoglobulin sequence,i.e., the analysis of residues that influence the ability of thecandidate immunoglobulin to bind its antigen. In this way, FR residuescan be selected and combined from the recipient and import sequences sothat the desired antibody characteristic, such as increased affinity forthe target antigen(s), is achieved. In general, the hypervariable regionresidues are directly and most substantially involved in influencingantigen binding.

As noted above, antibodies (or immunoglobulins) can be divided into fivedifferent classes, based on differences in the amino acid sequences inthe constant region of the heavy chains. All immunoglobulins within agiven class have very similar heavy chain constant regions. Thesedifferences can be detected by sequence studies or more commonly byserological means (i.e. by the use of antibodies directed to thesedifferences). Antibodies, or fragments thereof, of the present inventionmay be any class, and may, therefore, have a gamma, mu, alpha, delta, orepsilon heavy chain. A gamma chain may be gamma 1, gamma 2, gamma 3, orgamma 4; and an alpha chain may be alpha 1 or alpha 2.

In a preferred embodiment, an antibody of the present invention, orfragment thereof, is an IgG. IgG is considered the most versatileimmunoglobulin, because it is capable of carrying out all of thefunctions of immunoglobulin molecules. IgG is the major Ig in serum, andthe only class of Ig that crosses the placenta. IgG also fixescomplement, although the IgG4 subclass does not. Macrophages, monocytes,PMN's and some lymphocytes have Fc receptors for the Fc region of IgG.Not all subclasses bind equally well; IgG2 and IgG4 do not bind to Fcreceptors. A consequence of binding to the Fc receptors on PMN's,monocytes and macrophages is that the cell can now internalize theantigen better. IgG is an opsonin that enhances phagocytosis. Binding ofIgG to Fc receptors on other types of cells results in the activation ofother functions. Antibodies of the present invention may be of any IgGsubclass.

In another preferred embodiment, an antibody, or fragment thereof, ofthe present invention is an IgE. IgE is the least common serum Ig sinceit binds very tightly to Fc receptors on basophils and mast cells evenbefore interacting with antigen. As a consequence of its binding tobasophils and mast cells, IgE is involved in allergic reactions. Bindingof the allergen to the IgE on the cells results in the release ofvarious pharmacological mediators that result in allergic symptoms. IgEalso plays a role in parasitic helminth diseases. Eosinophils have Fcreceptors for IgE and binding of eosinophils to IgE-coated helminthsresults in killing of the parasite. IgE does not fix complement.

In various embodiments, antibodies of the present invention, andfragments thereof, comprise a variable light chain that is either kappaor lambda. The lamba chain may be any of subtype, including, e.g.,lambda 1, lambda 2, lambda 3, and lambda 4.

As noted above, the present invention further provides antibodyfragments comprising a polypeptide of the present invention. In certaincircumstances there are advantages of using antibody fragments, ratherthan whole antibodies. For example, the smaller size of the fragmentsallows for rapid clearance, and may lead to improved access to certaintissues, such as solid tumors. Examples of antibody fragments include:Fab, Fab′, F(ab′)₂ and Fv fragments; diabodies; linear antibodies;single-chain antibodies; and multispecific antibodies formed fromantibody fragments.

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., Journal ofBiochemical and Biophysical Methods 24:107-117 (1992); and Brennan etal., Science, 229:81 (1985)). However, these fragments can now beproduced directly by recombinant host cells. Fab, Fv and ScFv antibodyfragments can all be expressed in and secreted from E. coli, thusallowing the facile production of large amounts of these fragments.Fab′-SH fragments can be directly recovered from E. coli and chemicallycoupled to form F(ab′)₂ fragments (Carter et al., Bio/Technology10:163-167 (1992)). According to another approach, F(ab′), fragments canbe isolated directly from recombinant host cell culture. Fab and F(ab′)₂fragment with increased in vivo half-life comprising a salvage receptorbinding epitope residues are described in U.S. Pat. No. 5,869,046. Othertechniques for the production of antibody fragments will be apparent tothe skilled practitioner.

In other embodiments, the antibody of choice is a single chain Fvfragment (scFv). See WO 93/16185; U.S. Pat. Nos. 5,571,894; and5,587,458. Fv and sFv are the only species with intact combining sitesthat are devoid of constant regions. Thus, they are suitable for reducednonspecific binding during in vivo use. sFv fusion proteins may beconstructed to yield fusion of an effector protein at either the aminoor the carboxy terminus of an sFv. See Antibody Engineering, ed.Borrebaeck, supra. The antibody fragment may also be a “linearantibody”, e.g., as described in U.S. Pat. No. 5,641,870 for example.Such linear antibody fragments may be monospecific or bispecific.

In certain embodiments, antibodies of the present invention arebispecific or multispecific. Bispecific antibodies are antibodies thathave binding specificities for at least two different epitopes.Exemplary bispecific antibodies may bind to two different epitopes of asingle antigen. Other such antibodies may combine a first antigenbinding site with a binding site for a second antigen. Alternatively, ananti-HIV1 arm may be combined with an arm that binds to a triggeringmolecule on a leukocyte, such as a T-cell receptor molecule (e.g., CD3),or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) andFcγRIII (CD16), so as to focus and localize cellular defense mechanismsto the infected cell. Bispecific antibodies may also be used to localizecytotoxic agents to infected cells. These antibodies possess anHIV1-binding arm and an arm that binds the cytotoxic agent (e.g.,saporin, anti-interferon-α, vinca alkaloid, ricin A chain, methotrexateor radioactive isotope hapten). Bispecific antibodies can be prepared asfull length antibodies or antibody fragments (e.g., F(ab′)₂ bispecificantibodies). WO 96/16673 describes a bispecific anti-ErbB2/anti-FcγRIIIantibody and U.S. Pat. No. 5,837,234 discloses a bispecificanti-ErbB2/anti-FcγRI antibody. A bispecific anti-ErbB2/Fcα antibody isshown in WO98/02463. U.S. Pat. No. 5,821,337 teaches a bispecificanti-ErbB2/anti-CD3 antibody.

Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe co-expression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (Millstein et al.,Nature, 305:537-539 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture often different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829, and in Traunecker et al., EMBOJ., 10:3655-3659 (1991).

According to a different approach, antibody variable regions with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. Preferably, thefusion is with an Ig heavy chain constant domain, comprising at leastpart of the hinge, C_(H)2, and C_(H)3 regions. It is preferred to havethe first heavy-chain constant region (C_(H)1) containing the sitenecessary for light chain bonding, present in at least one of thefusions. DNAs encoding the immunoglobulin heavy chain fusions and, ifdesired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable host cell.This provides for greater flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yield of the desired bispecific antibody. It is,however, possible to insert the coding sequences for two or all threepolypeptide chains into a single expression vector when the expressionof at least two polypeptide chains in equal ratios results in highyields or when the ratios have no significant affect on the yield of thedesired chain combination.

In a preferred embodiment of this approach, the bispecific antibodiesare composed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation. This approach is disclosed in WO 94/04690. For furtherdetails of generating bispecific antibodies see, for example, Suresh etal., Methods in Enzymology, 121:210 (1986).

According to another approach described in U.S. Pat. No. 5,731,168, theinterface between a pair of antibody molecules can be engineered tomaximize the percentage of heterodimers that are recovered fromrecombinant cell culture. The preferred interface comprises at least apart of the C_(H) 3 domain. In this method, one or more small amino acidside chains from the interface of the first antibody molecule arereplaced with larger side chains (e.g., tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g., alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science, 229: 81 (1985) describe a procedure wherein intact antibodiesare proteolytically cleaved to generate F(ab′)₂ fragments. Thesefragments are reduced in the presence of the dithiol complexing agent,sodium arsenite, to stabilize vicinal dithiols and preventintermolecular disulfide formation. The Fab′ fragments generated arethen converted to thionitrobenzoate (TNB) derivatives. One of theFab′-TNB derivatives is then reconverted to the Fab′-thiol by reductionwith mercaptoethylamine and is mixed with an equimolar amount of theother Fab′-TNB derivative to form the bispecific antibody. Thebispecific antibodies produced can be used as agents for the selectiveimmobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describethe production of a humanized bispecific antibody F(ab′)₂ molecule. EachFab′ fragment was separately secreted from E. coli and subjected todirected chemical coupling in vitro to form the bispecific antibody. Thebispecific antibody thus formed was able to bind to cells overexpressingthe ErbB2 receptor and normal human T cells, as well as trigger thelytic activity of human cytotoxic lymphocytes against human breast tumortargets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise a V_(H)connected to a V_(L) by a linker that is too short to allow pairingbetween the two domains on the same chain. Accordingly, the V_(H) andV_(L) domains of one fragment are forced to pair with the complementaryV_(L) and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (sFv) dimers has also beenreported. See Gruber et al., J. Immunol., 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147: 60(1991). A multivalent antibody may be internalized (and/or catabolized)faster than a bivalent antibody by a cell expressing an antigen to whichthe antibodies bind. The antibodies of the present invention can bemultivalent antibodies with three or more antigen binding sites (e.g.,tetravalent antibodies), which can be readily produced by recombinantexpression of nucleic acid encoding the polypeptide chains of theantibody. The multivalent antibody can comprise a dimerization domainand three or more antigen binding sites. The preferred dimerizationdomain comprises (or consists of) an Fc region or a hinge region. Inthis scenario, the antibody will comprise an Fc region and three or moreantigen binding sites amino-terminal to the Fc region. The preferredmultivalent antibody herein comprises (or consists of) three to abouteight, but preferably four, antigen binding sites. The multivalentantibody comprises at least one polypeptide chain (and preferably twopolypeptide chains), wherein the polypeptide chain(s) comprise two ormore variable regions. For instance, the polypeptide chain(s) maycomprise VD1-(X1)_(n)-VD2-(X2)_(n)-Fc, wherein VD1 is a first variableregion, VD2 is a second variable region, Fc is one polypeptide chain ofan Fc region, X1 and X2 represent an amino acid or polypeptide, and n is0 or 1. For instance, the polypeptide chain(s) may comprise:VH-CH1-flexible linker-VH-CH1-Fc region chain; or VH-CH1-VH-CH1-Fcregion chain. The multivalent antibody herein preferably furthercomprises at least two (and preferably four) light chain variable regionpolypeptides. The multivalent antibody herein may, for instance,comprise from about two to about eight light chain variable regionpolypeptides. The light chain variable region polypeptides contemplatedhere comprise a light chain variable region and, optionally, furthercomprise a C_(L) domain.

Antibodies of the invention further include single chain antibodies. Inparticular embodiments, antibodies of the invention are internalizingantibodies.

Amino acid sequence modification(s) of the antibodies described hereinare contemplated. For example, it may be desirable to improve thebinding affinity and/or other biological properties of the antibody.Amino acid sequence variants of the antibody may be prepared byintroducing appropriate nucleotide changes into a polynucleotide thatencodes the antibody, or a chain thereof, or by peptide synthesis. Suchmodifications include, for example, deletions from, and/or insertionsinto and/or substitutions of, residues within the amino acid sequencesof the antibody. Any combination of deletion, insertion, andsubstitution may be made to arrive at the final antibody, provided thatthe final construct possesses the desired characteristics. The aminoacid changes also may alter post-translational processes of theantibody, such as changing the number or position of glycosylationsites. Any of the variations and modifications described above forpolypeptides of the present invention may be included in antibodies ofthe present invention.

A useful method for identification of certain residues or regions of anantibody that are preferred locations for mutagenesis is called “alaninescanning mutagenesis” as described by Cunningham and Wells in Science,244:1081-1085 (1989). Here, a residue or group of target residues areidentified (e.g., charged residues such as arg, asp, his, lys, and glu)and replaced by a neutral or negatively charged amino acid (mostpreferably alanine or polyalanine) to affect the interaction of theamino acids with PSCA antigen. Those amino acid locations demonstratingfunctional sensitivity to the substitutions then are refined byintroducing further or other variants at, or for, the sites ofsubstitution. Thus, while the site for introducing an amino acidsequence variation is predetermined, the nature of the mutation per seneed not be predetermined. For example, to analyze the performance of amutation at a given site, ala scanning or random mutagenesis isconducted at the target codon or region and the expressed anti-antibodyvariants are screened for the desired activity.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue or the antibody fusedto a cytotoxic polypeptide. Other insertional variants of an antibodyinclude the fusion to the N- or C-terminus of the antibody to an enzyme(e.g., for ADEPT) or a polypeptide that increases the serum half-life ofthe antibody.

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the antibody moleculereplaced by a different residue. The sites of greatest interest forsubstitutional mutagenesis include the hypervariable regions, but FRalterations are also contemplated. Conservative and non-conservativesubstitutions are contemplated.

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain.

Any cysteine residue not involved in maintaining the proper conformationof the antibody also may be substituted, generally with serine, toimprove the oxidative stability of the molecule and prevent aberrantcrosslinking. Conversely, cysteine bond(s) may be added to the antibodyto improve its stability (particularly where the antibody is an antibodyfragment such as an Fv fragment).

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody. Generally, theresulting variant(s) selected for further development will have improvedbiological properties relative to the parent antibody from which theyare generated. A convenient way for generating such substitutionalvariants involves affinity maturation using phage display. Briefly,several hypervariable region sites (e.g., 6-7 sites) are mutated togenerate all possible amino substitutions at each site. The antibodyvariants thus generated are displayed in a monovalent fashion fromfilamentous phage particles as fusions to the gene III product of M13packaged within each particle. The phage-displayed variants are thenscreened for their biological activity (e.g., binding affinity) asherein disclosed. In order to identify candidate hypervariable regionsites for modification, alanine scanning mutagenesis can be performed toidentify hypervariable region residues contributing significantly toantigen binding. Alternatively, or additionally, it may be beneficial toanalyze a crystal structure of the antigen-antibody complex to identifycontact points between the antibody and an antigen or infected cell.Such contact residues and neighboring residues are candidates forsubstitution according to the techniques elaborated herein. Once suchvariants are generated, the panel of variants is subjected to screeningas described herein and antibodies with superior properties in one ormore relevant assays may be selected for further development.

Another type of amino acid variant of the antibody alters the originalglycosylation pattern of the antibody. By altering is meant deleting oneor more carbohydrate moieties found in the antibody, and/or adding oneor more glycosylation sites that are not present in the antibody.Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used. Addition of glycosylation sites to theantibody is conveniently accomplished by altering the amino acidsequence such that it contains one or more of the above-describedtripeptide sequences (for N-linked glycosylation sites). The alterationmay also be made by the addition of, or substitution by, one or moreserine or threonine residues to the sequence of the original antibody(for O-linked glycosylation sites).

The antibody of the invention is modified with respect to effectorfunction, e.g., so as to enhance antigen-dependent cell-mediatedcyotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC) of theantibody. This may be achieved by introducing one or more amino acidsubstitutions in an Fc region of the antibody. Alternatively oradditionally, cysteine residue(s) may be introduced in the Fc region,thereby allowing interchain disulfide bond formation in this region. Thehomodimeric antibody thus generated may have improved internalizationcapability and/or increased complement-mediated cell killing andantibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J.Exp Med. 176:1191-1195 (1992) and Shopes, B. J. Immunol. 148:2918-2922(1992). Homodimeric antibodies with enhanced anti-infection activity mayalso be prepared using heterobifunctional cross-linkers as described inWolff et al., Cancer Research 53:2560-2565 (1993). Alternatively, anantibody can be engineered which has dual Fc regions and may therebyhave enhanced complement lysis and ADCC capabilities. See Stevenson etal., Anti-Cancer Drug Design 3:219-230 (1989). To increase the serumhalf-life of the antibody, one may incorporate a salvage receptorbinding epitope into the antibody (especially an antibody fragment) asdescribed in U.S. Pat. No. 5,739,277, for example. As used herein, theterm “salvage receptor binding epitope” refers to an epitope of the Fcregion of an IgG molecule (e.g., IgG₁, IgG₂, IgG₃, or IgG₄) that isresponsible for increasing the in vivo serum half-life of the IgGmolecule.

Antibodies of the present invention may also be modified to include anepitope tag or label, e.g., for use in purification or diagnosticapplications. The invention also pertains to therapy withimmunoconjugates comprising an antibody conjugated to an anti-canceragent such as a cytotoxic agent or a growth inhibitory agent.Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above.

Conjugates of an antibody and one or more small molecule toxins, such asa calicheamicin, maytansinoids, a trichothene, and CC1065, and thederivatives of these toxins that have toxin activity, are alsocontemplated herein.

In one preferred embodiment, an antibody (full length or fragments) ofthe invention is conjugated to one or more maytansinoid molecules.Maytansinoids are mitototic inhibitors that act by inhibiting tubulinpolymerization. Maytansine was first isolated from the east Africanshrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it wasdiscovered that certain microbes also produce maytansinoids, such asmaytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042).Synthetic maytansinol and derivatives and analogues thereof aredisclosed, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870;4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268;4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348;4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and4,371,533.

In an attempt to improve their therapeutic index, maytansine andmaytansinoids have been conjugated to antibodies specifically binding totumor cell antigens. Immunoconjugates containing maytansinoids and theirtherapeutic use are disclosed, for example, in U.S. Pat. Nos. 5,208,020,5,416,064 and European Patent EP 0 425 235 B1. Liu et al., Proc. Natl.Acad. Sci. USA 93:8618-8623 (1996) described immunoconjugates comprisinga maytansinoid designated DM1 linked to the monoclonal antibody C242directed against human colorectal cancer. The conjugate was found to behighly cytotoxic towards cultured colon cancer cells, and showedantitumor activity in an in vivo tumor growth assay.

Antibody-maytansinoid conjugates are prepared by chemically linking anantibody to a maytansinoid molecule without significantly diminishingthe biological activity of either the antibody or the maytansinoidmolecule. An average of 3-4 maytansinoid molecules conjugated perantibody molecule has shown efficacy in enhancing cytotoxicity of targetcells without negatively affecting the function or solubility of theantibody, although even one molecule of toxin/antibody would be expectedto enhance cytotoxicity over the use of naked antibody. Maytansinoidsare well known in the art and can be synthesized by known techniques orisolated from natural sources. Suitable maytansinoids are disclosed, forexample, in U.S. Pat. No. 5,208,020 and in the other patents andnonpatent publications referred to hereinabove. Preferred maytansinoidsare maytansinol and maytansinol analogues modified in the aromatic ringor at other positions of the maytansinol molecule, such as variousmaytansinol esters.

There are many linking groups known in the art for making antibodyconjugates, including, for example, those disclosed in U.S. Pat. No.5,208,020 or EP Patent 0 425 235 B1, and Chari et al., Cancer Research52: 127-131 (1992). The linking groups include disufide groups,thioether groups, acid labile groups, photolabile groups, peptidaselabile groups, or esterase labile groups, as disclosed in theabove-identified patents, disulfide and thioether groups beingpreferred.

Immunoconjugates may be made using a variety of bifunctional proteincoupling agents such as N-succinimidyl-3-(2-pyridyldithio)propionate(SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-carboxylate,iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCL), active esters (such as disuccinimidylsuberate), aldehydes (such as glutareldehyde), bis-azido compounds (suchas bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). Particularly preferred coupling agentsinclude N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) (Carlsson etal., Biochem. J. 173:723-737 [1978]) andN-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for adisulfide linkage. For example, a ricin immunotoxin can be prepared asdescribed in Vitetta et al., Science 238: 1098 (1987). Carbon-14-labeled1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid(MX-DTPA) is an exemplary chelating agent for conjugation ofradionucleotide to the antibody. See WO94/11026. The linker may be a“cleavable linker” facilitating release of the cytotoxic drug in thecell. For example, an acid-labile linker, Cancer Research 52: 127-131(1992); U.S. Pat. No. 5,208,020) may be used.

Another immunoconjugate of interest comprises an antibody conjugated toone or more calicheamicin molecules. The calicheamicin family ofantibiotics are capable of producing double-stranded DNA breaks atsub-picomolar concentrations. For the preparation of conjugates of thecalicheamicin family, see U.S. Pat. Nos. 5,712,374, 5,714,586,5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, 5,877,296 (all toAmerican Cyanamid Company). Another drug that the antibody can beconjugated is QFA which is an antifolate. Both calicheamicin and QFAhave intracellular sites of action and do not readily cross the plasmamembrane. Therefore, cellular uptake of these agents through antibodymediated internalization greatly enhances their cytotoxic effects.

Examples of other agents that can be conjugated to the antibodies of theinvention include BCNU, streptozoicin, vincristine and 5-fluorouracil,the family of agents known collectively LL-E33288 complex described inU.S. Pat. Nos. 5,053,394, 5,770,710, as well as esperamicins (U.S. Pat.No. 5,877,296).

Enzymatically active toxins and fragments thereof that can be usedinclude, e.g., diphtheria A chain, nonbinding active fragments ofdiphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricinA chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin and the tricothecenes. See, for example, WO 93/21232.

The present invention further includes an immunoconjugate formed betweenan antibody and a compound with nucleolytic activity (e.g., aribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).

For selective destruction of infected cells, the antibody includes ahighly radioactive atom. A variety of radioactive isotopes are availablefor the production of radioconjugated anti-PSCA antibodies. Examplesinclude At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Rc¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹²and radioactive isotopes of Lu. When the conjugate is used fordiagnosis, it may comprise a radioactive atom for scintigraphic studies,for example tc^(99m) or I¹²³, or a spin label for nuclear magneticresonance (NMR) imaging (also known as magnetic resonance imaging, mri),such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13,nitrogen-15, oxygen-17, gadolinium, manganese or iron.

The radio- or other label is incorporated in the conjugate in knownways. For example, the peptide may be biosynthesized or may besynthesized by chemical amino acid synthesis using suitable amino acidprecursors involving, for example, fluorine-19 in place of hydrogen.Labels such as tc^(99m) or I¹²³, Re¹⁸⁶, Re¹⁸⁸ and In¹¹¹ can be attachedvia a cysteine residue in the peptide. Yttrium-90 can be attached via alysine residue. The IODOGEN method (Fraker et al. (1978) Biochem.Biophys. Res. Commun. 80: 49-57 can be used to incorporate iodine-123.“Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989)describes other methods in detail.

Alternatively, a fusion protein comprising the antibody and cytotoxicagent is made, e.g., by recombinant techniques or peptide synthesis. Thelength of DNA may comprise respective regions encoding the two portionsof the conjugate either adjacent one another or separated by a regionencoding a linker peptide which does not destroy the desired propertiesof the conjugate. The antibodies of the present invention are also usedin antibody dependent enzyme mediated prodrug therapy (ADET) byconjugating the antibody to a prodrug-activating enzyme which converts aprodrug (e.g., a peptidyl chemotherapeutic agent, see WO81/01145) to anactive anti-cancer drug (see, e.g., WO 88/07378 and U.S. Pat. No.4,975,278).

The enzyme component of the immunoconjugate useful for ADEPT includesany enzyme capable of acting on a prodrug in such a way so as to covertit into its more active, cytotoxic form. Enzymes that are useful in themethod of this invention include, but are not limited to, alkalinephosphatase useful for converting phosphate-containing prodrugs intofree drugs; arylsulfatase useful for converting sulfate-containingprodrugs into free drugs; cytosine deaminase useful for convertingnon-toxic 5-fluorocytosine into the anti-cancer drug, 5-fluorouracil;proteases, such as serratia protease, thermolysin, subtilisin,carboxypeptidases and cathepsins (such as cathepsins B and L), that areuseful for converting peptide-containing prodrugs into free drugs;D-alanylcarboxypeptidases, useful for converting prodrugs that containD-amino acid substituents; carbohydrate-cleaving enzymes such as3-galactosidase and neuraminidase useful for converting glycosylatedprodrugs into free drugs; 3-lactamase useful for converting drugsderivatized with β-lactams into free drugs; and penicillin amidases,such as penicillin V amidase or penicillin G amidase, useful forconverting drugs derivatized at their amine nitrogens with phenoxyacetylor phenylacetyl groups, respectively, into free drugs. Alternatively,antibodies with enzymatic activity, also known in the art as “abzymes”,can be used to convert the prodrugs of the invention into free activedrugs (see, e.g., Massey, Nature 328: 457-458 (1987)). Antibody-abzymeconjugates can be prepared as described herein for delivery of theabzyme to a infected cell population.

The enzymes of this invention can be covalently bound to the antibodiesby techniques well known in the art such as the use of theheterobifunctional crosslinking reagents discussed above. Alternatively,fusion proteins comprising at least the antigen binding region of anantibody of the invention linked to at least a functionally activeportion of an enzyme of the invention can be constructed usingrecombinant DNA techniques well known in the art (see, e.g., Neubergeret al., Nature, 312: 604-608 (1984).

Other modifications of the antibody are contemplated herein. Forexample, the antibody may be linked to one of a variety ofnonproteinaceous polymers, e.g., polyethylene glycol, polypropyleneglycol, polyoxyalkylenes, or copolymers of polyethylene glycol andpolypropylene glycol. The antibody also may be entrapped inmicrocapsules prepared, for example, by coacervation techniques or byinterfacial polymerization (for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate)microcapsules,respectively), in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules), or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed.,(1980).

The antibodies disclosed herein are also formulated as immunoliposomes.A “liposome” is a small vesicle composed of various types of lipids,phospholipids and/or surfactant that is useful for delivery of a drug toa mammal. The components of the liposome are commonly arranged in abilayer formation, similar to the lipid arrangement of biologicalmembranes. Liposomes containing the antibody are prepared by methodsknown in the art, such as described in Epstein et al., Proc. Natl. Acad.Sci. USA, 82:3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA,77:4030 (1980); U.S. Pat. Nos. 4,485,045 and 4,544,545; and WO97/38731published Oct. 23, 1997. Liposomes with enhanced circulation time aredisclosed in U.S. Pat. No. 5,013,556.

Particularly useful liposomes can be generated by the reverse phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desired adiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al., J. Biol.Chem. 257: 286-288 (1982) via a disulfide interchange reaction. Achemotherapeutic agent is optionally contained within the liposome. SeeGabizon et al., J. National Cancer Inst. 81(19)1484 (1989). Antibodiesof the present invention, or fragments thereof, may possess any of avariety of biological or functional characteristics. In certainembodiments, these antibodies are HIV1 protein specific antibodies,indicating that they specifically bind to or preferentially bind to HIV1as compared to a normal control cell.

In particular embodiments, an antibody of the present invention is anantagonist antibody, which partially or fully blocks or inhibits abiological activity of a polypeptide or cell to which it specifically orpreferentially binds. In other embodiments, an antibody of the presentinvention is a growth inhibitory antibody, which partially or fullyblocks or inhibits the growth of an infected cell to which it binds. Inanother embodiment, an antibody of the present invention inducesapoptosis. In yet another embodiment, an antibody of the presentinvention induces or promotes antibody-dependent cell-mediatedcytotoxicity or complement dependent cytotoxicity.

HIV1-expressing cells or virus described above are used to screen thebiological sample obtained from a patient infected with HIV1 for thepresence of antibodies that preferentially bind to the cell expressingHIV1 polypeptides using standard biological techniques. For example, incertain embodiments, the antibodies may be labeled, and the presence oflabel associated with the cell detected, e.g., using FMAT or FACsanalysis. In particular embodiments, the biological sample is blood,serum, plasma, bronchial lavage, or saliva. Methods of the presentinvention may be practiced using high throughput techniques.

Identified human antibodies may then be characterized further. Forexample the particular conformational epitopes with in the HIV1polypeptides that are necessary or sufficient for binding of theantibody may be determined, e.g., using site-directed mutagenesis ofexpressed HIV1 polypeptides. These methods may be readily adapted toidentify human antibodies that bind any protein expressed on a cellsurface. Furthermore, these methods may be adapted to determine bindingof the antibody to the virus itself, as opposed to a cell expressingrecombinant HIV1 or infected with the virus.

Polynucleotide sequences encoding the antibodies, variable regionsthereof, or antigen-binding fragments thereof may be subcloned intoexpression vectors for the recombinant production of human anti-HIV1antibodies. In one embodiment, this is accomplished by obtainingmononuclear cells from the patient from the serum containing theidentified HIV1 antibody was obtained; producing B cell clones from themononuclear cells; inducing the B cells to become antibody-producingplasma cells; and screening the supernatants produced by the plasmacells to determine if it contains the HIV1 antibody. Once a B cell clonethat produces an HIV1 antibody is identified, reverse-transcriptionpolymerase chain reaction (RT-PCR) is performed to clone the DNAsencoding the variable regions or portions thereof of the HIV1 antibody.These sequences are then subcloned into expression vectors suitable forthe recombinant production of human HIV1 antibodies. The bindingspecificity may be confirmed by determining the recombinant antibody'sability to bind cells expressing HIV1 polypeptide.

In particular embodiments of the methods described herein, B cellsisolated from peripheral blood or lymph nodes are sorted, e.g., based ontheir being CD19 positive, and plated, e.g., as low as a single cellspecificity per well, e.g., in 96, 384, or 1536 well configurations. Thecells are induced to differentiate into antibody-producing cells, e.g.,plasma cells, and the culture supernatants are harvested and tested forbinding to cells expressing the infectious agent polypeptide on theirsurface using, e.g., FMAT or FACS analysis. Positive wells are thensubjected to whole well RT-PCR to amplify heavy and light chain variableregions of the IgG molecule expressed by the clonal daughter plasmacells. The resulting PCR products encoding the heavy and light chainvariable regions, or portions thereof, are subcloned into human antibodyexpression vectors for recombinant expression. The resulting recombinantantibodies are then tested to confirm their original binding specificityand may be further tested for pan-specificity across various strains ofisolates of the infectious agent.

Thus, in one embodiment, a method of identifying HIV1 antibodies ispracticed as follows. First, full length or approximately full lengthHIV cDNAs are transfected into a cell line for expression of HIV1polypeptides. Secondly, individual human plasma or sera samples aretested for antibodies that bind the cell-expressed HIV1 polypeptides.And lastly, MAbs derived from plasma- or serum-positive individuals arecharacterized for binding to the same cell-expressed HIV1 polypeptides.Further definition of the fine specificities of the MAbs can beperformed at this point.

Polynucleotides that encode the HIV1 antibodies or portions thereof ofthe present invention may be isolated from cells expressing HIV1antibodies, according to methods available in the art and describedherein, including amplification by polymerase chain reaction usingprimers specific for conserved regions of human antibody polypeptides.For example, light chain and heavy chain variable regions may be clonedfrom the B cell according to molecular biology techniques described inWO 92/02551; U.S. Pat. No. 5,627,052; or Babcook et al., Proc. Natl.Acad. Sci. USA 93:7843-48 (1996). In certain embodiments,polynucleotides encoding all or a region of both the heavy and lightchain variable regions of the IgG molecule expressed by the clonaldaughter plasma cells expressing the HIV1 antibody are subcloned andsequenced. The sequence of the encoded polypeptide may be readilydetermined from the polynucleotide sequence.

Isolated polynucleotides encoding a polypeptide of the present inventionmay be subcloned into an expression vector to recombinantly produceantibodies and polypeptides of the present invention, using proceduresknown in the art and described herein.

Binding properties of an antibody (or fragment thereof) to HIV1polypeptides or HIv1 infected cells or tissues may generally bedetermined and assessed using immunodetection methods including, forexample, immunofluorescence-based assays, such as immuno-histochemistry(IHC) and/or fluorescence-activated cell sorting (FACS). Immunoassaymethods may include controls and procedures to determine whetherantibodies bind specifically to HIV1 polypeptides from one or morespecific clades or strains of HIV, and do not recognize or cross-reactwith normal control cells.

Following pre-screening of serum to identify patients that produceantibodies to an infectious agent or polypeptide thereof, e.g., HIV1,the methods of the present invention typically include the isolation orpurification of B cells from a biological sample previously obtainedfrom a patient or subject. The patient or subject may be currently orpreviously diagnosed with or suspect or having a particular disease orinfection, or the patient or subject may be considered free or aparticular disease or infection. Typically, the patient or subject is amammal and, in particular embodiments, a human. The biological samplemay be any sample that contains B cells, including but not limited to,lymph node or lymph node tissue, pleural effusions, peripheral blood,ascites, tumor tissue, or cerebrospinal fluid (CSF). In variousembodiments, B cells are isolated from different types of biologicalsamples, such as a biological sample affected by a particular disease orinfection. However, it is understood that any biological samplecomprising B cells may be used for any of the embodiments of the presentinvention.

Once isolated, the B cells are induced to produce antibodies, e.g., byculturing the B cells under conditions that support B cell proliferationor development into a plasmacyte, plasmablast, or plasma cell. Theantibodies are then screened, typically using high throughputtechniques, to identify an antibody that specifically binds to a targetantigen, e.g., a particular tissue, cell, infectious agent, orpolypeptide. In certain embodiments, the specific antigen, e.g., cellsurface polypeptide bound by the antibody is not known, while in otherembodiments, the antigen specifically bound by the antibody is known.

According to the present invention, B cells may be isolated from abiological sample, e.g., a tumor, tissue, peripheral blood or lymph nodesample, by any means known and available in the art. B cells aretypically sorted by FACS based on the presence on their surface of a Bcell-specific marker, e.g., CD19, CD138, and/or surface IgG. However,other methods known in the art may be employed, such as, e.g., columnpurification using CD19 magnetic beads or IgG-specific magnetic beads,followed by elution from the column. However, magnetic isolation of Bcells utilizing any marker may result in loss of certain B cells.Therefore, in certain embodiments, the isolated cells are not sortedbut, instead, phicol-purified mononuclear cells isolated from tumor aredirectly plated to the appropriate or desired number of specificitiesper well.

In order to identify B cells that produce an infectious agent-specificantibody, the B cells are typically plated at low density (e.g., asingle cell specificity per well, 1-10 cells per well, 10-100 cells perwell, 1-100 cells per well, less than 10 cells per well, or less than100 cells per well) in multi-well or microtiter plates, e.g., in 96,384, or 1536 well configurations. When the B cells are initially platedat a density greater than one cell per well, then the methods of thepresent invention may include the step of subsequently diluting cells ina well identified as producing an antigen-specific antibody, until asingle cell specificity per well is achieved, thereby facilitating theidentification of the B cell that produces the antigen-specificantibody. Cell supernatants or a portion thereof and/or cells may befrozen and stored for future testing and later recovery of antibodypolynucleotides.

In certain embodiments, the B cells are cultured under conditions thatfavor the production of antibodies by the B cells. For example, the Bcells may be cultured under conditions favorable for B cellproliferation and differentiation to yield antibody-producingplasmablast, plasmacytes, or plasma cells. In particular embodiments,the B cells are cultured in the presence of a B cell mitogen, such aslipopolysaccharide (LPS) or CD40 ligand. In one specific embodiment, Bcells are differentiated to antibody-producing cells by culturing themwith feed cells and/or other B cell activators, such as CD40 ligand.

Cell culture supernatants or antibodies obtained therefrom may be testedfor their ability to bind to a target antigen, using routine methodsavailable in the art, including those described herein. In particularembodiments, culture supernatants are tested for the presence ofantibodies that bind to a target antigen using high-throughput methods.For example, B cells may be cultured in multi-well microtiter dishes,such that robotic plate handlers may be used to simultaneously samplemultiple cell supernatants and test for the presence of antibodies thatbind to a target antigen. In particular embodiments, antigens are boundto beads, e.g., paramagnetic or latex beads) to facilitate the captureof antibody/antigen complexes. In other embodiments, antigens andantibodies are fluorescently labeled (with different labels) and FACSanalysis is performed to identify the presence of antibodies that bindto target antigen. In one embodiment, antibody binding is determinedusing FMAT™ analysis and instrumentation (Applied Biosystems, FosterCity, Calif.). FMAT™ is a fluorescence macro-confocal platform forhigh-throughput screening, which mix-and-read, non-radioactive assaysusing live cells or beads.

In the context of comparing the binding of an antibody to a particulartarget antigen (e.g., a biological sample such as infected tissue orcells, or infectious agents) as compared to a control sample (e.g., abiological sample such as uninfected cells, or a different infectiousagent), in various embodiments, the antibody is considered topreferentially bind a particular target antigen if at least two-fold, atleast three-fold, at least five-fold, or at least ten-fold more antibodybinds to the particular target antigen as compared to the amount thatbinds a control sample.

Polynucleotides encoding antibody chains, variable regions thereof, orfragments thereof, may be isolated from cells utilizing any meansavailable in the art. In one embodiment, polynucleotides are isolatedusing polymerase chain reaction (PCR), e.g., reverse transcription-PCR(RT-PCR) using oligonucleotide primers that specifically bind to heavyor light chain encoding polynucleotide sequences or complements thereofusing routine procedures available in the art. In one embodiment,positive wells are subjected to whole well RT-PCR to amplify the heavyand light chain variable regions of the IgG molecule expressed by theclonal daughter plasma cells. These PCR products may be sequenced.

The resulting PCR products encoding the heavy and light chain variableregions or portions thereof are then subcloned into human antibodyexpression vectors and recombinantly expressed according to routineprocedures in the art (see, e.g., U.S. Pat. No. 7,112,439). The nucleicacid molecules encoding a tumor-specific antibody or fragment thereof,as described herein, may be propagated and expressed according to any ofa variety of well-known procedures for nucleic acid excision, ligation,transformation, and transfection. Thus, in certain embodimentsexpression of an antibody fragment may be preferred in a prokaryotichost cell, such as Escherichia coli (see, e.g., Pluckthun et al.,Methods Enzymol. 178:497-515 (1989)). In certain other embodiments,expression of the antibody or an antigen-binding fragment thereof may bepreferred in a eukaryotic host cell, including yeast (e.g.,Saccharomyces cerevisiae, Schizosaccharonmyces pombe, and Pichiapastoris); animal cells (including mammalian cells); or plant cells.Examples of suitable animal cells include, but are not limited to,myeloma, COS, CHO, or hybridoma cells. Examples of plant cells includetobacco, corn, soybean, and rice cells. By methods known to those havingordinary skill in the art and based on the present disclosure, a nucleicacid vector may be designed for expressing foreign sequences in aparticular host system, and then polynucleotide sequences encoding thetumor-specific antibody (or fragment thereof) may be inserted. Theregulatory elements will vary according to the particular host.

One or more replicable expression vectors containing a polynucleotideencoding a variable and/or constant region may be prepared and used totransform an appropriate cell line, for example, a non-producing myelomacell line, such as a mouse NSO line or a bacterium, such as E. coli, inwhich production of the antibody will occur. In order to obtainefficient transcription and translation, the polynucleotide sequence ineach vector should include appropriate regulatory sequences,particularly a promoter and leader sequence operatively linked to thevariable region sequence. Particular methods for producing antibodies inthis way are generally well known and routinely used. For example,molecular biology procedures are described by Sambrook et al. (MolecularCloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory,New York, 1989; see also Sambrook et al., 3rd ed., Cold Spring HarborLaboratory, New York, (2001)). While not required, in certainembodiments, regions of polynucleotides encoding the recombinantantibodies may be sequenced. DNA sequencing can be performed asdescribed in Sanger et al. (Proc. Natl. Acad. Sci. USA 74:5463 (1977))and the Amersham International p1c sequencing handbook and includingimprovements thereto.

In particular embodiments, the resulting recombinant antibodies orfragments thereof are then tested to confirm their original specificityand may be further tested for pan-specificity, e.g., with relatedinfectious agents. In particular embodiments, an antibody identified orproduced according to methods described herein is tested for cellkilling via antibody dependent cellular cytotoxicity (ADCC) orapoptosis, and/or well as its ability to internalize.

The present invention, in other aspects, provides polynucleotidecompositions. In preferred embodiments, these polynucleotides encode apolypeptide of the invention, e.g., a region of a variable chain of anantibody that binds to HIV1. Polynucleotides of the invention aresingle-stranded (coding or antisense) or double-stranded DNA (genomic,cDNA or synthetic) or RNA molecules. RNA molecules include, but are notlimited to, HnRNA molecules, which contain introns and correspond to aDNA molecule in a one-to-one manner, and mRNA molecules, which do notcontain introns. Alternatively, or in addition, coding or non-codingsequences are present within a polynucleotide of the present invention.Also alternatively, or in addition, a polynucleotide is linked to othermolecules and/or support materials of the invention. Polynucleotides ofthe invention are used, e.g., in hybridization assays to detect thepresence of an HIV1 antibody in a biological sample, and in therecombinant production of polypeptides of the invention. Further, theinvention includes all polynucleotides that encode any polypeptide ofthe present invention.

In other related embodiments, the invention provides polynucleotidevariants having substantial identity to the sequences of 1443_C16,1456_P20, 1460_G14, 1495_C14 or 1496_C09, for example those comprisingat least 70% sequence identity, preferably at least 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, or 99% or higher, sequence identity compared to apolynucleotide sequence of this invention, as determined using themethods described herein, (e.g., BLAST analysis using standardparameters). One skilled in this art will recognize that these valuescan be appropriately adjusted to determine corresponding identity ofproteins encoded by two nucleotide sequences by taking into accountcodon degeneracy, amino acid similarity, reading frame positioning, andthe like.

Typically, polynucleotide variants contain one or more substitutions,additions, deletions and/or insertions, preferably such that theimmunogenic binding properties of the polypeptide encoded by the variantpolynucleotide is not substantially diminished relative to a polypeptideencoded by a polynucleotide sequence specifically set forth herein.

In additional embodiments, the present invention provides polynucleotidefragments comprising various lengths of contiguous stretches of sequenceidentical to or complementary to one or more of the sequences disclosedherein. For example, polynucleotides are provided by this invention thatcomprise at least about 10, 15, 20, 30, 40, 50, 75, 100, 150, 200, 300,400, 500 or 1000 or more contiguous nucleotides of one or more of thesequences disclosed herein as well as all intermediate lengths therebetween. As used herein, the term “intermediate lengths” is meant todescribe any length between the quoted values, such as 16, 17, 18, 19,etc.; 21, 22, 23, etc.; 30, 31, 32, etc.; 50, 51, 52, 53, etc.; 100,101, 102, 103, etc.; 150, 151, 152, 153, etc.; including all integersthrough 200-500; 500-1,000, and the like.

In another embodiment of the invention, polynucleotide compositions areprovided that are capable of hybridizing under moderate to highstringency conditions to a polynucleotide sequence provided herein, or afragment thereof, or a complementary sequence thereof. Hybridizationtechniques are well known in the art of molecular biology. For purposesof illustration, suitable moderately stringent conditions for testingthe hybridization of a polynucleotide of this invention with otherpolynucleotides include prewashing in a solution of 5×SSC, 0.5% SDS, 1.0mM EDTA (pH 8.0); hybridizing at 50° C.-60° C., 5×SSC, overnight;followed by washing twice at 65° C. for 20 minutes with each of 2×, 0.5×and 0.2×SSC containing 0.1% SDS. One skilled in the art will understandthat the stringency of hybridization can be readily manipulated, such asby altering the salt content of the hybridization solution and/or thetemperature at which the hybridization is performed. For example, inanother embodiment, suitable highly stringent hybridization conditionsinclude those described above, with the exception that the temperatureof hybridization is increased, e.g., to 60-65° C. or 65-70° C.

In preferred embodiments, the polypeptide encoded by the polynucleotidevariant or fragment has the same binding specificity (i.e., specificallyor preferentially binds to the same epitope or HIV strain) as thepolypeptide encoded by the native polynucleotide. In certain preferredembodiments, the polynucleotides described above, e.g., polynucleotidevariants, fragments and hybridizing sequences, encode polypeptides thathave a level of binding activity of at least about 50%, preferably atleast about 70%, and more preferably at least about 90% of that for apolypeptide sequence specifically set forth herein.

The polynucleotides of the present invention, or fragments thereof,regardless of the length of the coding sequence itself, may be combinedwith other DNA sequences, such as promoters, polyadenylation signals,additional restriction enzyme sites, multiple cloning sites, othercoding segments, and the like, such that their overall length may varyconsiderably. A nucleic acid fragment of almost any length is employed,with the total length preferably being limited by the ease ofpreparation and use in the intended recombinant DNA protocol. Forexample, illustrative polynucleotide segments with total lengths ofabout 10,000, about 5000, about 3000, about 2,000, about 1,000, about500, about 200, about 100, about 50 base pairs in length, and the like,(including all intermediate lengths) are included in manyimplementations of this invention.

It will be appreciated by those of ordinary skill in the art that, as aresult of the degeneracy of the genetic code, there are multiplenucleotide sequences that encode a polypeptide as described herein. Someof these polynucleotides bear minimal homology to the nucleotidesequence of any native gene. Nonetheless, polynucleotides that encode apolypeptide of the present invention but which vary due to differencesin codon usage are specifically contemplated by the invention. Further,alleles of the genes including the polynucleotide sequences providedherein are within the scope of the invention. Alleles are endogenousgenes that are altered as a result of one or more mutations, such asdeletions, additions and/or substitutions of nucleotides. The resultingmRNA and protein may, but need not, have an altered structure orfunction. Alleles may be identified using standard techniques (such ashybridization, amplification and/or database sequence comparison).

In certain embodiments of the present invention, mutagenesis of thedisclosed polynucleotide sequences is performed in order to alter one ormore properties of the encoded polypeptide, such as its bindingspecificity or binding strength. Techniques for mutagenesis arewell-known in the art, and are widely used to create variants of bothpolypeptides and polynucleotides. A mutagenesis approach, such assite-specific mutagenesis, is employed for the preparation of variantsand/or derivatives of the polypeptides described herein. By thisapproach, specific modifications in a polypeptide sequence are madethrough mutagenesis of the underlying polynucleotides that encode them.These techniques provides a straightforward approach to prepare and testsequence variants, for example, incorporating one or more of theforegoing considerations, by introducing one or more nucleotide sequencechanges into the polynucleotide.

Site-specific mutagenesis allows the production of mutants through theuse of specific oligonucleotide sequences include the nucleotidesequence of the desired mutation, as well as a sufficient number ofadjacent nucleotides, to provide a primer sequence of sufficient sizeand sequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Mutations are employed in a selectedpolynucleotide sequence to improve, alter, decrease, modify, orotherwise change the properties of the polynucleotide itself, and/oralter the properties, activity, composition, stability, or primarysequence of the encoded polypeptide.

In other embodiments of the present invention, the polynucleotidesequences provided herein are used as probes or primers for nucleic acidhybridization, e.g., as PCR primers. The ability of such nucleic acidprobes to specifically hybridize to a sequence of interest enables themto detect the presence of complementary sequences in a given sample.However, other uses are also encompassed by the invention, such as theuse of the sequence information for the preparation of mutant speciesprimers, or primers for use in preparing other genetic constructions. Assuch, nucleic acid segments of the invention that include a sequenceregion of at least about a 15-nucleotide long contiguous sequence thathas the same sequence as, or is complementary to, a 15 nucleotide longcontiguous sequence disclosed herein is particularly useful. Longercontiguous identical or complementary sequences, e.g., those of about20, 30, 40, 50, 100, 200, 500, 1000 (including all intermediate lengths)including full length sequences, and all lengths in between, are alsoused in certain embodiments.

Polynucleotide molecules having sequence regions consisting ofcontiguous nucleotide stretches of 10-14, 15-20, 30, 50, or even of100-200 nucleotides or so (including intermediate lengths as well),identical or complementary to a polynucleotide sequence disclosedherein, are particularly contemplated as hybridization probes for usein, e.g., Southern and Northern blotting, and/or primers for use in,e.g., polymerase chain reaction (PCR). The total size of fragment, aswell as the size of the complementary stretch (es), ultimately dependson the intended use or application of the particular nucleic acidsegment. Smaller fragments are generally used in hybridizationembodiments, wherein the length of the contiguous complementary regionmay be varied, such as between about 15 and about 100 nucleotides, butlarger contiguous complementarity stretches may be used, according tothe length complementary sequences one wishes to detect.

The use of a hybridization probe of about 15-25 nucleotides in lengthallows the formation of a duplex molecule that is both stable andselective. Molecules having contiguous complementary sequences overstretches greater than 12 bases in length are generally preferred,though, in order to increase stability and selectivity of the hybrid,and thereby improve the quality and degree of specific hybrid moleculesobtained. Nucleic acid molecules having gene-complementary stretches of15 to 25 contiguous nucleotides, or even longer where desired, aregenerally preferred.

Hybridization probes are selected from any portion of any of thesequences disclosed herein. All that is required is to review thesequences set forth herein, or to any continuous portion of thesequences, from about 15-25 nucleotides in length up to and includingthe full length sequence, that one wishes to utilize as a probe orprimer. The choice of probe and primer sequences is governed by variousfactors. For example, one may wish to employ primers from towards thetermini of the total sequence.

Polynucleotide of the present invention, or fragments or variantsthereof, are readily prepared by, for example, directly synthesizing thefragment by chemical means, as is commonly practiced using an automatedoligonucleotide synthesizer. Also, fragments are obtained by applicationof nucleic acid reproduction technology, such as the PCR™ technology ofU.S. Pat. No. 4,683,202, by introducing selected sequences intorecombinant vectors for recombinant production, and by other recombinantDNA techniques generally known to those of skill in the art of molecularbiology.

The invention provides vectors and host cells comprising a nucleic acidof the present invention, as well as recombinant techniques for theproduction of a polypeptide of the present invention. Vectors of theinvention include those capable of replication in any type of cell ororganism, including, e.g., plasmids, phage, cosmids, and minichromosomes. In various embodiments, vectors comprising a polynucleotideof the present invention are vectors suitable for propagation orreplication of the polynucleotide, or vectors suitable for expressing apolypeptide of the present invention. Such vectors are known in the artand commercially available.

Polynucleotides of the present invention are synthesized, whole or inparts that are then combined, and inserted into a vector using routinemolecular and cell biology techniques, including, e.g., subcloning thepolynucleotide into a linearized vector using appropriate restrictionsites and restriction enzymes. Polynucleotides of the present inventionare amplified by polymerase chain reaction using oligonucleotide primerscomplementary to each strand of the polynucleotide. These primers alsoinclude restriction enzyme cleavage sites to facilitate subcloning intoa vector. The replicable vector components generally include, but arenot limited to, one or more of the following: a signal sequence, anorigin of replication, and one or more marker or selectable genes.

In order to express a polypeptide of the present invention, thenucleotide sequences encoding the polypeptide, or functionalequivalents, are inserted into an appropriate expression vector, i.e., avector that contains the necessary elements for the transcription andtranslation of the inserted coding sequence. Methods well known to thoseskilled in the art are used to construct expression vectors containingsequences encoding a polypeptide of interest and appropriatetranscriptional and translational control elements. These methodsinclude in vitro recombinant DNA techniques, synthetic techniques, andin vivo genetic recombination. Such techniques are described, forexample, in Sambrook, J., et al. (1989) Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. etal. (1989) Current Protocols in Molecular Biology, John Wiley & Sons,New York. N.Y.

A variety of expression vector/host systems are utilized to contain andexpress polynucleotide sequences. These include, but are not limited to,microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems.

Within one embodiment, the variable regions of a gene expressing amonoclonal antibody of interest are amplified from a hybridoma cellusing nucleotide primers. These primers are synthesized by one ofordinary skill in the art, or may be purchased from commerciallyavailable sources (see, e.g., Stratagene (La Jolla, Calif.), which sellsprimers for amplifying mouse and human variable regions. The primers areused to amplify heavy or light chain variable regions, which are theninserted into vectors such as ImmunoZAP™ H or ImmunoZAP™ L (Stratagene),respectively. These vectors are then introduced into E. coli, yeast, ormammalian-based systems for expression. Large amounts of a single-chainprotein containing a fusion of the V_(H) and V_(L) domains are producedusing these methods (see Bird et al., Science 242:423-426 (1988)).

The “control elements” or “regulatory sequences” present in anexpression vector are those non-translated regions of the vector, e.g.,enhancers, promoters, 5′ and 3′ untranslated regions, that interact withhost cellular proteins to carry out transcription and translation. Suchelements may vary in their strength and specificity. Depending on thevector system and host utilized, any number of suitable transcriptionand translation elements, including constitutive and induciblepromoters, are used.

Examples of promoters suitable for use with prokaryotic hosts includethe phoa promoter, β-lactamase and lactose promoter systems, alkalinephosphatase promoter, a tryptophan (trp) promoter system, and hybridpromoters such as the tac promoter. However, other known bacterialpromoters are suitable. Promoters for use in bacterial systems alsousually contain a Shine-Dalgarno sequence operably linked to the DNAencoding the polypeptide. Inducible promoters such as the hybrid lacZpromoter of the PBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) orPSPORT1 plasmid (Gibco BRL, Gaithersburg, Md.) and the like are used.

A variety of promoter sequences are known for eukaryotes and any areused according to the present invention. Virtually all eukaryotic geneshave an AT-rich region located approximately 25 to 30 bases upstreamfrom the site where transcription is initiated. Another sequence found70 to 80 bases upstream from the start of transcription of many genes isa CNCAAT region where N may be any nucleotide. At the 3′ end of mosteukaryotic genes is an AATAAA sequence that may be the signal foraddition of the poly A tail to the 3′ end of the coding sequence. All ofthese sequences are suitably inserted into eukaryotic expressionvectors.

In mammalian cell systems, promoters from mammalian genes or frommammalian viruses are generally preferred. Polypeptide expression fromvectors in mammalian host cells are controlled, for example, bypromoters obtained from the genomes of viruses such as polyoma virus,fowlpox virus, adenovirus (e.g., Adenovirus 2), bovine papilloma virus,avian sarcoma virus, cytomegalovirus (CMV), a retrovirus, hepatitis-Bvirus and most preferably Simian Virus 40 (SV40), from heterologousmammalian promoters, e.g., the actin promoter or an immunoglobulinpromoter, and from heat-shock promoters, provided such promoters arecompatible with the host cell systems. If it is necessary to generate acell line that contains multiple copies of the sequence encoding apolypeptide, vectors based on SV40 or EBV may be advantageously usedwith an appropriate selectable marker. One example of a suitableexpression vector is pcDNA-3.1 (Invitrogen, Carlsbad, Calif.), whichincludes a CMV promoter.

A number of viral-based expression systems are available for mammalianexpression of polypeptides. For example, in cases where an adenovirus isused as an expression vector, sequences encoding a polypeptide ofinterest may be ligated into an adenovirus transcription/translationcomplex consisting of the late promoter and tripartite leader sequence.Insertion in a non-essential E1 or E3 region of the viral genome may beused to obtain a viable virus that is capable of expressing thepolypeptide in infected host cells (Logan, J. and Shenk, T. (1984) Proc.Natl. Acad. Sci. 81:3655-3659). In addition, transcription enhancers,such as the Rous sarcoma virus (RSV) enhancer, may be used to increaseexpression in mammalian host cells.

In bacterial systems, any of a number of expression vectors are selecteddepending upon the use intended for the expressed polypeptide. Forexample, when large quantities are desired, vectors that direct highlevel expression of fusion proteins that are readily purified are used.Such vectors include, but are not limited to, the multifunctional E.coli cloning and expression vectors such as BLUESCRIPT (Stratagene), inwhich the sequence encoding the polypeptide of interest may be ligatedinto the vector in frame with sequences for the amino-terminal Met andthe subsequent 7 residues of β-galactosidase, so that a hybrid proteinis produced; pIN vectors (Van Heeke, G. and S. M. Schuster (1989) J.Biol. Chem. 264:5503-5509); and the like. pGEX Vectors (Promega,Madison, Wis.) are also used to express foreign polypeptides as fusionproteins with glutathione S-transferase (GST). In general, such fusionproteins are soluble and can easily be purified from lysed cells byadsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. Proteins made in such systems are designedto include heparin, thrombin, or factor XA protease cleavage sites sothat the cloned polypeptide of interest can be released from the GSTmoiety at will.

In the yeast, Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase, and PGH are used. Examples of other suitable promoter sequencesfor use with yeast hosts include the promoters for 3-phosphoglyceratekinase or other glycolytic enzymes, such as enolase,glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. For reviews, see Ausubel etal. (supra) and Grant et al. (1987) Methods Enzymol. 153:516-544. Otheryeast promoters that are inducible promoters having the additionaladvantage of transcription controlled by growth conditions include thepromoter regions for alcohol dehydrogenase 2, isocytochrome C, acidphosphatase, degradative enzymes associated with nitrogen metabolism,metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymesresponsible for maltose and galactose utilization. Suitable vectors andpromoters for use in yeast expression are further described in EP73,657. Yeast enhancers also are advantageously used with yeastpromoters.

In cases where plant expression vectors are used, the expression ofsequences encoding polypeptides are driven by any of a number ofpromoters. For example, viral promoters such as the 35S and 19Spromoters of CaMV are used alone or in combination with the omega leadersequence from TMV (Takamatsu, N. (1987) EMBO J. 6:307-311.Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters are used (Coruzzi, G. et al. (1984) EMBO J.3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter,J., et al. (1991) Results Probl. Cell Differ. 17:85-105). Theseconstructs can be introduced into plant cells by direct DNAtransformation or pathogen-mediated transfection. Such techniques aredescribed in a number of generally available reviews (see, e.g., Hobbs,S. or Murry, L. E. in McGraw Hill Yearbook of Science and Technology(1992) McGraw Hill, New York, N.Y.; pp. 191-196).

An insect system is also used to express a polypeptide of interest. Forexample, in one such system, Autographa californica nuclear polyhedrosisvirus (AcNPV) is used as a vector to express foreign genes in Spodopterafrugiperda cells or in Trichoplusia larvae. The sequences encoding thepolypeptide are cloned into a non-essential region of the virus, such asthe polyhedrin gene, and placed under control of the polyhedrinpromoter. Successful insertion of the polypeptide-encoding sequencerenders the polyhedrin gene inactive and produce recombinant viruslacking coat protein. The recombinant viruses are then used to infect,for example, S. frugiperda cells or Trichoplusia larvae, in which thepolypeptide of interest is expressed (Engelhard, E. K. et al. (1994)Proc. Natl. Acad. Sci. 91:3224-3227).

Specific initiation signals are also used to achieve more efficienttranslation of sequences encoding a polypeptide of interest. Suchsignals include the ATG initiation codon and adjacent sequences. Incases where sequences encoding the polypeptide, its initiation codon,and upstream sequences are inserted into the appropriate expressionvector, no additional transcriptional or translational control signalsmay be needed. However, in cases where only coding sequence, or aportion thereof, is inserted, exogenous translational control signalsincluding the ATG initiation codon are provided. Furthermore, theinitiation codon is in the correct reading frame to ensure correcttranslation of the inserted polynucleotide. Exogenous translationalelements and initiation codons are of various origins, both natural andsynthetic.

Transcription of a DNA encoding a polypeptide of the invention is oftenincreased by inserting an enhancer sequence into the vector. Manyenhancer sequences are known, including, e.g., those identified in genesencoding globin, elastase, albumin, α-fetoprotein, and insulin.Typically, however, an enhancer from a eukaryotic cell virus is used.Examples include the SV40 enhancer on the late side of the replicationorigin (bp 100-270), the cytomegalovirus early promoter enhancer, thepolyoma enhancer on the late side of the replication origin, andadenovirus enhancers. See also Yaniv, Nature 297:17-18 (1982) onenhancing elements for activation of eukaryotic promoters. The enhanceris spliced into the vector at a position 5′ or 3′ to thepolypeptide-encoding sequence, but is preferably located at a site 5′from the promoter.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) typically also contain sequences necessary for thetermination of transcription and for stabilizing the mRNA. Suchsequences are commonly available from the 5′ and, occasionally 3′,untranslated regions of eukaryotic or viral DNAs or cDNAs. These regionscontain nucleotide segments transcribed as polyadenylated fragments inthe untranslated portion of the mRNA encoding anti-PSCA antibody. Oneuseful transcription termination component is the bovine growth hormonepolyadenylation region. See WO94/11026 and the expression vectordisclosed therein.

Suitable host cells for cloning or expressing the DNA in the vectorsherein are the prokaryote, yeast, plant or higher eukaryote cellsdescribed above. Examples of suitable prokaryotes for this purposeinclude eubacteria, such as Gram-negative or Gram-positive organisms,for example, Enterobacteriaceae such as Escherichia, e.g., E. coli,Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonellatyphimurium, Serratia, e.g., Serratia marcescans, and Shigella, as wellas Bacilli such as B. subtilis and B. licheniformis (e.g., B.licheniformis 41P disclosed in DD 266,710 published 12 Apr. 1989),Pseudomonas such as P. aeruginosa, and Streptomyces. One preferred E.coli cloning host is E. coli 294 (ATCC 31,446), although other strainssuch as E. coli B, E. coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC27,325) are suitable. These examples are illustrative rather thanlimiting.

Saccharomyces cerevisiae, or common baker's yeast, is the most commonlyused among lower eukaryotic host microorganisms. However, a number ofother genera, species, and strains are commonly available and usedherein, such as Schizosaccharomyces pombe; Kluyveromyces hosts such as,e.g., K lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045),K wickeramii (ATCC 24,178). K. waltii (ATCC 56,500), K. drosophilarum(ATCC 36,906), K. thermotolerans, and K. marxianus; yarrowia (EP402,226); Pichia pastoris. (EP 183,070); Candida; Trichodernma reesia(EP 244,234); Neurospora crassa; Schwanniomnyces such as Schwanniomycesoccidentalis; and filamentous fungi such as, e.g., Neurospora,Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulansand A. niger.

In certain embodiments, a host cell strain is chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing that cleaves a “prepro” form of theprotein is also used to facilitate correct insertion, folding and/orfunction. Different host cells such as CHO, COS, HeLa, MDCK, HEK293, andWI38, which have specific cellular machinery and characteristicmechanisms for such post-translational activities, are chosen to ensurethe correct modification and processing of the foreign protein.

Methods and reagents specifically adapted for the expression ofantibodies or fragments thereof are also known and available in the art,including those described, e.g., in U.S. Pat. Nos. 4,816,567 and6,331,415. In various embodiments, antibody heavy and light chains, orfragments thereof, are expressed from the same or separate expressionvectors. In one embodiment, both chains are expressed in the same cell,thereby facilitating the formation of a functional antibody or fragmentthereof.

Full length antibody, antibody fragments, and antibody fusion proteinsare produced in bacteria, in particular when glycosylation and Fceffector function are not needed, such as when the therapeutic antibodyis conjugated to a cytotoxic agent (e.g., a toxin) and theimmunoconjugate by itself shows effectiveness in infected celldestruction. For expression of antibody fragments and polypeptides inbacteria, see, e.g., U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523,which describes translation initiation region (TIR) and signal sequencesfor optimizing expression and secretion. After expression, the antibodyis isolated from the E. coli cell paste in a soluble fraction and can bepurified through, e.g., a protein A or G column depending on theisotype. Final purification can be carried out using a process similarto that used for purifying antibody expressed e.g., in CHO cells.

Suitable host cells for the expression of glycosylated polypeptides andantibodies are derived from multicellular organisms. Examples ofinvertebrate cells include plant and insect cells. Numerous baculoviralstrains and variants and corresponding permissive insect host cells fromhosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti(mosquito), Aedes albopicius (mosquito), Drosophila melanogaster(fruitfly), and Bombyx mori have been identified. A variety of viralstrains for transfection are publicly available, e.g., the L-1 variantof Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV,and such viruses are used as the virus herein according to the presentinvention, particularly for transfection of Spodoptera frugiperda cells.Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,and tobacco are also utilized as hosts.

Methods of propagation of antibody polypeptides and fragments thereof invertebrate cells in culture (tissue culture) are encompassed by theinvention. Examples of mammalian host cell lines used in the methods ofthe invention are monkey kidney CV1 line transformed by SV40 (COS-7,ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subclonedfor growth in suspension culture, Graham et al., J. Gen Virol. 36:59(1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamsterovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod.23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African greenmonkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinomacells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138,ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor(MMT 060562, ATCC CCL51); TR1 cells (Mather et al., Annals N.Y. Acad.Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatomaline (Hep G2).

Host cells are transformed with the above-described expression orcloning vectors for polypeptide production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.

For long-term, high-yield production of recombinant proteins, stableexpression is generally preferred. For example, cell lines that stablyexpress a polynucleotide of interest are transformed using expressionvectors that contain viral origins of replication and/or endogenousexpression elements and a selectable marker gene on the same or on aseparate vector. Following the introduction of the vector, cells areallowed to grow for 1-2 days in an enriched media before they areswitched to selective media. The purpose of the selectable marker is toconfer resistance to selection, and its presence allows growth andrecovery of cells that successfully express the introduced sequences.Resistant clones of stably transformed cells are proliferated usingtissue culture techniques appropriate to the cell type.

A plurality of selection systems are used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler, M. et al. (1977) Cell 11:223-32) and adeninephosphoribosyltransferase (Lowy, I. et al. (1990) Cell 22:817-23) genesthat are employed in tk⁻ or aprt⁻ cells, respectively. Also,antimetabolite, antibiotic or herbicide resistance is used as the basisfor selection; for example, dhfr, which confers resistance tomethotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.77:3567-70); npt, which confers resistance to the aminoglycosides,neomycin and G-418 (Colbere-Garapin, F. et al. (1981) J. Mol. Biol.150:1-14); and als or pat, which confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively (Murry, supra).Additional selectable genes have been described. For example, trpBallows cells to utilize indole in place of tryptophan, and hisD allowscells to utilize histinol in place of histidine (Hartman, S. C. and R.C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51). The use ofvisible markers has gained popularity with such markers as anthocyanins,beta-glucuronidase and its substrate GUS, and luciferase and itssubstrate luciferin, being widely used not only to identifytransformants, but also to quantify the amount of transient or stableprotein expression attributable to a specific vector system (Rhodes, C.A. et al. (1995) Methods Mol. Biol. 55:121-131).

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, its presence and expression isconfirmed. For example, if the sequence encoding a polypeptide isinserted within a marker gene sequence, recombinant cells containingsequences are identified by the absence of marker gene function.Alternatively, a marker gene is placed in tandem with apolypeptide-encoding sequence under the control of a single promoter.Expression of the marker gene in response to induction or selectionusually indicates expression of the tandem gene as well.

Alternatively, host cells that contain and express a desiredpolynucleotide sequence are identified by a variety of procedures knownto those of skill in the art. These procedures include, but are notlimited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay orimmunoassay techniques which include, for example, membrane, solution,or chip based technologies for the detection and/or quantification ofnucleic acid or protein.

A variety of protocols for detecting and measuring the expression ofpolynucleotide-encoded products, using either polyclonal or monoclonalantibodies specific for the product are known in the art. Nonlimitingexamples include enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS).A two-site, monoclonal-based immunoassay utilizing monoclonal antibodiesreactive to two non-interfering epitopes on a given polypeptide ispreferred for some applications, but a competitive binding assay mayalso be employed. These and other assays are described, among otherplaces, in Hampton, R. et al. (1990; Serological Methods, a LaboratoryManual, APS Press, St Paul. Minn.) and Maddox, D. E. et al. (1983; J.Exp. Med. 158:1211-1216).

Various labels and conjugation techniques are known by those skilled inthe art and are used in various nucleic acid and amino acid assays.Means for producing labeled hybridization or PCR probes for detectingsequences related to polynucleotides include oligolabeling, nicktranslation, end-labeling or PCR amplification using a labelednucleotide. Alternatively, the sequences, or any portions thereof arecloned into a vector for the production of an mRNA probe. Such vectorsare known in the art, are commercially available, and are used tosynthesize RNA probes in vitro by addition of an appropriate RNApolymerase such as T7, T3, or SP6 and labeled nucleotides. Theseprocedures are conducted using a variety of commercially available kits.Suitable reporter molecules or labels, which are used include, but arenot limited to, radionucleotides, enzymes, fluorescent,chemiluminescent, or chromogenic agents as well as substrates,cofactors, inhibitors, magnetic particles, and the like.

The polypeptide produced by a recombinant cell is secreted or containedintracellularly depending on the sequence and/or the vector used.Expression vectors containing polynucleotides of the invention aredesigned to contain signal sequences that direct secretion of theencoded polypeptide through a prokaryotic or eukaryotic cell membrane.

In certain embodiments, a polypeptide of the invention is produced as afusion polypeptide further including a polypeptide domain thatfacilitates purification of soluble proteins. Suchpurification-facilitating domains include, but are not limited to, metalchelating peptides such as histidine-tryptophan modules that allowpurification on immobilized metals, protein A domains that allowpurification on immobilized immunoglobulin, and the domain utilized inthe FLAGS extension/affinity purification system (Amgen, Seattle,Wash.). The inclusion of cleavable linker sequences such as thosespecific for Factor XA or enterokinase (Invitrogen. San Diego, Calif.)between the purification domain and the encoded polypeptide are used tofacilitate purification. An exemplary expression vector provides forexpression of a fusion protein containing a polypeptide of interest anda nucleic acid encoding 6 histidine residues preceding a thioredoxin oran enterokinase cleavage site. The histidine residues facilitatepurification on IMIAC (immobilized metal ion affinity chromatography) asdescribed in Porath, J. et al. (1992, Prot. Exp. Purif. 3:263-281) whilethe enterokinase cleavage site provides a means for purifying thedesired polypeptide from the fusion protein. A discussion of vectorsused for producing fusion proteins is provided in Kroll, D. J. et al.(1993; DNA Cell Biol. 12:441-453).

In certain embodiments, a polypeptide of the present invention is fusedwith a heterologous polypeptide, which may be a signal sequence or otherpolypeptide having a specific cleavage site at the N-terminus of themature protein or polypeptide. The heterologous signal sequence selectedpreferably is one that is recognized and processed (i.e., cleaved by asignal peptidase) by the host cell. For prokaryotic host cells, thesignal sequence is selected, for example, from the group of the alkalinephosphatase, penicillinase, 1pp, or heat-stable enterotoxin II leaders.For yeast secretion, the signal sequence is selected from, e.g., theyeast invertase leader, a factor leader (including Saccharomyces andKluyveromyces a factor leaders), or acid phosphatase leader, the C.albicans glucoamylase leader, or the signal described in WO 90/13646. Inmammalian cell expression, mammalian signal sequences as well as viralsecretory leaders, for example, the herpes simplex gD signal, areavailable.

When using recombinant techniques, the polypeptide or antibody isproduced intracellularly, in the periplasmic space, or directly secretedinto the medium. If the polypeptide or antibody is producedintracellularly, as a first step, the particulate debris, either hostcells or lysed fragments, are removed, for example, by centrifugation orultrafiltration. Carter et al., Bio/Technology 10:163-167 (1992)describe a procedure for isolating antibodies that are secreted to theperiplasmic space of E. coli. Briefly, cell paste is thawed in thepresence of sodium acetate (pH 3.5), EDTA, andphenylmethylsulfonylfluoride (PMSF) over about 30 min. Cell debris isremoved by centrifugation. Where the polypeptide or antibody is secretedinto the medium, supernatants from such expression systems are generallyfirst concentrated using a commercially available protein concentrationfilter, for example, an Amicon or Millipore Pellicon ultrafiltrationunit. Optionally, a protease inhibitor such as PMSF is included in anyof the foregoing steps to inhibit proteolysis and antibiotics areincluded to prevent the growth of adventitious contaminants.

The polypeptide or antibody composition prepared from the cells arepurified using, for example, hydroxylapatite chromatography, gelelectrophoresis, dialysis, and affinity chromatography, with affinitychromatography being the preferred purification technique. Thesuitability of protein A as an affinity ligand depends on the speciesand isotype of any immunoglobulin Fc domain that is present in thepolypeptide or antibody. Protein A is used to purify antibodies orfragments thereof that are based on human γ₁, γ₂, or γ₄ heavy chains(Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G isrecommended for all mouse isotypes and for human γ₃ (Guss et al., EMBOJ. 5:15671575 (1986)). The matrix to which the affinity ligand isattached is most often agarose, but other matrices are available.Mechanically stable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where thepolypeptide or antibody comprises a C_(H) 3 domain, the Bakerbond ABX™resin (J. T. Baker, Phillipsburg, N.J.) is useful for purification.Other techniques for protein purification such as fractionation on anion-exchange column, ethanol precipitation, Reverse Phase HPLC,chromatography on silica, chromatography on heparin SEPHAROSE™chromatography on an anion or cation exchange resin (such as apolyaspartic acid column), chromatofocusing, SDS-PAGE, and ammoniumsulfate precipitation are also available depending on the polypeptide orantibody to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe polypeptide or antibody of interest and contaminants are subjectedto low pH hydrophobic interaction chromatography using an elution bufferat a pH between about 2.5-4.5, preferably performed at low saltconcentrations (e.g., from about 0-0.25M salt).

The invention further includes pharmaceutical formulations including apolypeptide, antibody, or modulator of the present invention, at adesired degree of purity, and a pharmaceutically acceptable carrier,excipient, or stabilizer (Remington's Pharmaceutical Sciences 16thedition, Osol, A. Ed. (1980)). In certain embodiments, pharmaceuticalformulations are prepared to enhance the stability of the polypeptide orantibody during storage, e.g., in the form of lyophilized formulationsor aqueous solutions.

Acceptable carriers, excipients, or stabilizers are nontoxic torecipients at the dosages and concentrations employed, and include,e.g., buffers such as acetate. Tris, phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA;tonicifiers such as trehalose and sodium chloride; sugars such assucrose, mannitol, trehalose or sorbitol; surfactant such aspolysorbate; salt-forming counter-ions such as sodium; metal complexes(e.g. Zn-protein complexes); and/or non-ionic surfactants such asTWEEN™, PLURONICS™ or polyethylene glycol (PEG). In certain embodiments,the therapeutic formulation preferably comprises the polypeptide orantibody at a concentration of between 5-200 mg/ml, preferably between10-100 mg/ml.

The formulations herein also contain one or more additional therapeuticagents suitable for the treatment of the particular indication, e.g.,infection being treated, or to prevent undesired side-effects.Preferably, the additional therapeutic agent has an activitycomplementary to the polypeptide or antibody of the resent invention,and the two do not adversely affect each other. For example, in additionto the polypeptide or antibody of the invention, an additional or secondantibody, anti-viral agent, anti-infective agent and/or cardioprotectantis added to the formulation. Such molecules are suitably present in thepharmaceutical formulation in amounts that are effective for the purposeintended.

The active ingredients, e.g., polypeptides and antibodies of theinvention and other therapeutic agents, are also entrapped inmicrocapsules prepared, for example, by coacervation techniques or byinterfacial polymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and polymethylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Sustained-release preparations are prepared. Suitable examples ofsustained-release preparations include, but are not limited to,semi-permeable matrices of solid hydrophobic polymers containing theantibody, which matrices are in the form of shaped articles, e.g.,films, or microcapsules. Nonlimiting examples of sustained-releasematrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxyburyric acid.

Formulations to be used for in vivo administration are preferablysterile. This is readily accomplished by filtration through sterilefiltration membranes.

Antibodies of the invention can be coupled to a drug for delivery to atreatment site or coupled to a detectable label to facilitate imaging ofa site comprising cells of interest, such as cells infected with HIV.Methods for coupling antibodies to drugs and detectable labels are wellknown in the art, as are methods for imaging using detectable labels.Labeled antibodies may be employed in a wide variety of assays,employing a wide variety of labels. Detection of the formation of anantibody-antigen complex between an antibody of the invention and anepitope of interest (an HIV epitope) can be facilitated by attaching adetectable substance to the antibody. Suitable detection means includethe use of labels such as radionucleotides, enzymes, coenzymes,fluorescers, chemiluminescers, chromogens, enzyme substrates orco-factors, enzyme inhibitors, prosthetic group complexes, freeradicals, particles, dyes, and the like. Examples of suitable enzymesinclude horseradish peroxidase, alkaline phosphatase, β-galactosidase,or acetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material isluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S, or ³H. Such labeled reagents may be used in avariety of well-known assays, such as radioimmunoassays, enzymeimmunoassays, e.g., ELISA, fluorescent immunoassays, and the like.

The antibodies are tagged with such labels by known methods. Forinstance, coupling agents such as aldehydes, carbodiimides, dimaleimide,imidates, succinimides, bid-diazotized benzadine and the like are usedto tag the antibodies with the above-described fluorescent,chemiluminescent, and enzyme labels. An enzyme is typically combinedwith an antibody using bridging molecules such as carbodiimides,periodate, diisocyanates, glutaraldehyde and the like. Various labelingtechniques are described in Morrison, Methods in Enzymology 32b, 103(1974), Syvanen et al., J. Biol. Chem. 284, 3762 (1973) and Bolton andHunter, Biochem J. 133, 529(1973).

An antibody according to the invention may be conjugated to atherapeutic moiety such as a cytotoxin, a therapeutic agent, or aradioactive metal ion or radioisotope. Examples of radioisotopesinclude, but are not limited to, I-131, I-123, I-125, Y-90, Re-188,Re-186, At-211, Cu-67, Bi-212, Bi-213, Pd-109, Tc-99, In-111, and thelike. Such antibody conjugates can be used for modifying a givenbiological response; the drug moiety is not to be construed as limitedto classical chemical therapeutic agents. For example, the drug moietymay be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, a toxin such as abrin,ricin A, pseudomonas exotoxin, or diphtheria toxin.

Techniques for conjugating such therapeutic moiety to antibodies arewell known. See, for example, Arnon et al. (1985) “Monoclonal Antibodiesfor Immunotargeting of Drugs in Cancer Therapy,” in MonoclonalAntibodies and Cancer Therapy, ed. Reisfeld et al. (Alan R. Liss, Inc.),pp. 243-256; ed. Hellstrom et al. (1987) “Antibodies for Drug Delivery,”in Controlled Drug Delivery, ed. Robinson et al. (2d ed; Marcel Dekker,Inc.), pp. 623-653; Thorpe (1985) “Antibody Carriers of Cytotoxic Agentsin Cancer Therapy: A Review,” in Monoclonal Antibodies '84: Biologicaland Clinical Applications, ed. Pinchera et al. pp. 475-506 (EditriceKurtis, Milano, Italy, 1985); “Analysis, Results, and Future Prospectiveof the Therapeutic Use of Radiolabeled Antibody in Cancer Therapy,” inMonoclonal Antibodies for Cancer Detection and Therapy, ed. Baldwin etal. (Academic Press, New York, 1985), pp. 303-316; and Thorpe et al.(1982) Immunol. Rev. 62:119-158.

Diagnostic methods generally involve contacting a biological sampleobtained from a patient, such as, e.g., blood, serum, saliva, urine,sputum, a cell swab sample, or a tissue biopsy, with an HIV1 antibodyand determining whether the antibody preferentially binds to the sampleas compared to a control sample or predetermined cut-off value, therebyindicating the presence of infected cells. In particular embodiments, atleast two-fold, three-fold, or five-fold more HIV1 antibody binds to aninfected cell as compared to an appropriate control normal cell ortissue sample. A pre-determined cut-off value is determined, e.g., byaveraging the amount of HIV1 antibody that binds to several differentappropriate control samples under the same conditions used to performthe diagnostic assay of the biological sample being tested.

Bound antibody is detected using procedures described herein and knownin the art. In certain embodiments, diagnostic methods of the inventionare practiced using HIV1 antibodies that are conjugated to a detectablelabel, e.g., a fluorophore, to facilitate detection of bound antibody.However, they are also practiced using methods of secondary detection ofthe HIV1 antibody. These include, for example, RIA, ELISA,precipitation, agglutination, complement fixation andimmuno-fluorescence.

HIV1 antibodies of the present invention are capable of differentiatingbetween patients with and patients without an HIV infection, anddetermining whether or not a patient has an infection, using therepresentative assays provided herein. According to one method, abiological sample is obtained from a patient suspected of having orknown to have HIV1 infection. In preferred embodiments, the biologicalsample includes cells from the patient. The sample is contacted with anHIV1 antibody, e.g., for a time and under conditions sufficient to allowthe HIV1 antibody to bind to infected cells present in the sample. Forinstance, the sample is contacted with an HIV1 antibody for 10 seconds,30 seconds, 1 minute, 5 minutes, 10 minutes, 30 minutes, 1 hour, 6hours, 12 hours, 24 hours, 3 days or any point in between. The amount ofbound HIV1 antibody is determined and compared to a control value, whichmay be, e.g., a pre-determined value or a value determined from normaltissue sample. An increased amount of antibody bound to the patientsample as compared to the control sample is indicative of the presenceof infected cells in the patient sample.

In a related method, a biological sample obtained from a patient iscontacted with an HIV1 antibody for a time and under conditionssufficient to allow the antibody to bind to infected cells. Boundantibody is then detected, and the presence of bound antibody indicatesthat the sample contains infected cells. This embodiment is particularlyuseful when the HIV1 antibody does not bind normal cells at a detectablelevel.

Different HIV1 antibodies possess different binding and specificitycharacteristics. Depending upon these characteristics, particular HIV1antibodies are used to detect the presence of one or more strains ofHIV1. For example, certain antibodies bind specifically to only one orseveral strains of HIV1, whereas others bind to all or a majority ofdifferent strains of HIV1. Antibodies specific for only one strain ofHIV1 are used to identify the strain of an infection.

In certain embodiments, antibodies that bind to an infected cellpreferably generate a signal indicating the presence of an infection inat least about 20% of patients with the infection being detected, morepreferably at least about 30% of patients. Alternatively, or inaddition, the antibody generates a negative signal indicating theabsence of the infection in at least about 90% of individuals withoutthe infection being detected. Each antibody satisfies the abovecriteria; however, antibodies of the present invention are used incombination to improve sensitivity.

The present invention also includes kits useful in performing diagnosticand prognostic assays using the antibodies of the present invention.Kits of the invention include a suitable container comprising an HIV1antibody of the invention in either labeled or unlabeled form. Inaddition, when the antibody is supplied in a labeled form suitable foran indirect binding assay, the kit further includes reagents forperforming the appropriate indirect assay. For example, the kit includesone or more suitable containers including enzyme substrates orderivatizing agents, depending on the nature of the label. Controlsamples and/or instructions are also included.

Passive immunization has proven to be an effective and safe strategy forthe prevention and treatment of viral diseases. (See Keller et al.,Clin. Microbiol. Rev. 13:602-14 (2000); Casadevall, Nat. Biotechnol.20:114 (2002); Shibata et al., Nat. Med. 5:204-10 (1999); and Igarashiet al., Nat. Med. 5:211-16 (1999), each of which are incorporated hereinby reference)). Passive immunization using human monoclonal antibodies,provide an immediate treatment strategy for emergency prophylaxis andtreatment of HIV1.

HIV1 antibodies and fragments thereof, and therapeutic compositions, ofthe invention specifically bind or preferentially bind to infectedcells, as compared to normal control uninfected cells and tissue. Thus,these HIV1 antibodies are used to selectively target infected cells ortissues in a patient, biological sample, or cell population. In light ofthe infection-specific binding properties of these antibodies, thepresent invention provides methods of regulating (e.g., inhibiting) thegrowth of infected cells, methods of killing infected cells, and methodsof inducing apoptosis of infected cells. These methods includecontacting an infected cell with an HIV1 antibody of the invention.These methods are practiced in vitro, ex vivo, and in vivo.

In various embodiments, antibodies of the invention are intrinsicallytherapeutically active. Alternatively, or in addition, antibodies of theinvention are conjugated to a cytotoxic agent or growth inhibitoryagent, e.g., a radioisotope or toxin that is used in treating infectedcells bound or contacted by the antibody.

Subjects at risk for HIV1-related diseases or disorders include patientswho have come into contact with an infected person or who have beenexposed to HIV1 in some other way. Administration of a prophylacticagent can occur prior to the manifestation of symptoms characteristic ofHIV1-related disease or disorder, such that a disease or disorder isprevented or, alternatively, delayed in its progression.

Methods for preventing an increase in HIV1 virus titer, virusreplication, virus proliferation or an amount of an HIV1 viral proteinin a subject are further provided. In one embodiment, a method includesadministering to the subject an amount of an HIV1 antibody effective toprevent an increase in HIV1 titer, virus replication or an amount of anHIV1 protein of one or more HIV strains or isolates in the subject.

For in vivo treatment of human and non-human patients, the patient isusually administered or provided a pharmaceutical formulation includingan HIV1 antibody of the invention. When used for in vivo therapy, theantibodies of the invention are administered to the patient intherapeutically effective amounts (i.e., amounts that eliminate orreduce the patient's viral burden). The antibodies are administered to ahuman patient, in accord with known methods, such as intravenousadministration, e.g., as a bolus or by continuous infusion over a periodof time, by intramuscular, intraperitoneal, intracerobrospinal,subcutaneous, intra-articular, intrasynovial, intrathecal, oral,topical, or inhalation routes. The antibodies may be administeredparenterally, when possible, at the target cell site, or intravenously.Intravenous or subcutaneous administration of the antibody is preferredin certain embodiments. Therapeutic compositions of the invention areadministered to a patient or subject systemically, parenterally, orlocally.

For parenteral administration, the antibodies are formulated in a unitdosage injectable form (solution, suspension, emulsion) in associationwith a pharmaceutically acceptable, parenteral vehicle. Examples of suchvehicles are water, saline, Ringer's solution, dextrose solution, and 5%human serum albumin. Nonaqueous vehicles such as fixed oils and ethyloleate are also used. Liposomes are used as carriers. The vehiclecontains minor amounts of additives such as substances that enhanceisotonicity and chemical stability, e.g., buffers and preservatives. Theantibodies are typically formulated in such vehicles at concentrationsof about 1 mg/ml to 10 mg/ml.

The dose and dosage regimen depends upon a variety of factors readilydetermined by a physician, such as the nature of the infection and thecharacteristics of the particular cytotoxic agent or growth inhibitoryagent conjugated to the antibody (when used), e.g., its therapeuticindex, the patient, and the patient's history. Generally, atherapeutically effective amount of an antibody is administered to apatient. In particular embodiments, the amount of antibody administeredis in the range of about 0.1 mg/kg to about 50 mg/kg of patient bodyweight. Depending on the type and severity of the infection, about 0.1mg/kg to about 50 mg/kg body weight (e.g., about 0.1-15 mg/kg/dose) ofantibody is an initial candidate dosage for administration to thepatient, whether, for example, by one or more separate administrations,or by continuous infusion. The progress of this therapy is readilymonitored by conventional methods and assays and based on criteria knownto the physician or other persons of skill in the art.

In one particular embodiment, an immunoconjugate including the antibodyconjugated with a cytotoxic agent is administered to the patient.Preferably, the immunoconjugate is internalized by the cell, resultingin increased therapeutic efficacy of the immunoconjugate in killing thecell to which it binds. In one embodiment, the cytotoxic agent targetsor interferes with the nucleic acid in the infected cell. Examples ofsuch cytotoxic agents are described above and include, but are notlimited to, maytansinoids, calicheamicins, ribonucleases and DNAendonucleases.

Other therapeutic regimens are combined with the administration of theHIV1 antibody of the present invention. The combined administrationincludes co-administration, using separate formulations or a singlepharmaceutical formulation, and consecutive administration in eitherorder, wherein preferably there is a time period while both (or all)active agents simultaneously exert their biological activities.Preferably such combined therapy results in a synergistic therapeuticeffect.

In certain embodiments, it is desirable to combine administration of anantibody of the invention with another antibody directed against anotherantigen associated with the infectious agent.

Aside from administration of the antibody protein to the patient, theinvention provides methods of administration of the antibody by genetherapy. Such administration of nucleic acid encoding the antibody isencompassed by the expression “administering a therapeutically effectiveamount of an antibody”. See, for example, PCT Patent ApplicationPublication WO96/07321 concerning the use of gene therapy to generateintracellular antibodies.

In another embodiment, anti-HIV1 antibodies of the invention are used todetermine the structure of bound antigen, e.g., conformational epitopes,the structure of which is then used to develop a vaccine having ormimicking this structure, e.g., through chemical modeling and SARmethods. Such a vaccine could then be used to prevent HIV1 infection.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

EXAMPLES Example 1 Selection of Patient Sample

Serum from approximately 1,800 HIV-1 infected donors from Asia,Australia, Europe, North America and sub-Saharan African countries werescreened for neutralization activity and donors who exhibit among thebroadest and most potent neutralizing serum activity observed to datewere identified. (Simek, M. D., J Virol (2009)). Monoclonal antibodieswere generated from these donors using different approaches.

A patient was selected based upon the patient's eligibility forenrollment, which was defined as: male or female at least 18 years ofage with documented HIV infection for at least three years, clinicallyasymptomatic at the time of enrollment, and not currently receivingantiretroviral therapy. (Simek, M. D., J Virol (2009 July)83(14):7337-48). Selection of individuals for monoclonal antibodygeneration was based on a rank-order high throughput analyticalscreening algorithm. The volunteer was identified as an individual withbroad neutralizing serum based on broad and potent neutralizing activityagainst a cross-clade pseudovirus panel.

A novel high-throughput strategy was used to screen IgG-containingculture supernatants from approximately 30,000 activated memory B cellsfrom a clade A infected donor for recombinant, monomeric gp120_(JR-CSF)and gp41_(HxB2) (Env) binding as well as neutralization activity againstHIV-1_(JR-CSF) and HIV-1_(SF162) as shown in Table 1. The memory B cellswere cultured at near clonal density such that the authentic antibodyheavy and light chain pair could be reconstituted from each culturewell.

Example 2 Generation of Monoclonal Antibodies

The human monoclonal antibody discovery platform utilized a short term Bcell culture system to interrogate the memory B cell repertoire. 30,300CD19⁺ and surface IgG-expressing memory B cells were isolated from tenmillion peripheral blood mononuclear cells (PBMC) of the HIV-1 infecteddonor. CD19⁺/sIgG⁺ B cells were then seeded in 384-well microtiterplates at an average of 1.3 cells/well under conditions that promoted Bcell activation, proliferation, terminal differentiation and antibodysecretion. Culture supernatants were screened in a high throughputformat for binding reactivity to recombinant gp120 and gp41 indirectlyand directly immobilized on ELISA plates, respectively. In parallel, theculture supernatants were also screened for neutralization activity in ahigh throughput micro-neutralization assay.

Heavy and light variable regions were isolated from lysates of selectedneutralizing hits by RT-PCR amplification using family-specific primersets. From positive family-specific PCR reactions, pools of the VH orVL-region clones were cloned into an expression vector upstream to humanIgG1 constant domain sequence. Minipreps (QIAGEN, Valencia, Calif.) ofthese DNA pools, derived from suspension bacterial cultures, werecombined in all possible heavy and light chain family-specific pairs andused to transiently transfect 293 cells. All transfectant supernatantscontaining secreted recombinant antibodies were screened in ELISA andneutralization assays. For B-cell wells that contained more than one Bcell clone per culture well, multiple VH and VL domain sequences wereisolated. ELISA (for B-cell wells positive for ELISA) and neutralizationscreens identified the heavy and light chain combination pools thatreconstituted the binding and neutralizing activity as observed for theB-cell well. DNA sequences of the heavy and light chain variable regionsfor all neutralizing mAbs were confirmed by multiple sequencingreactions using purified DNA from maxipreps (QIAGEN).

Example 3 Screening of Monoclonal Antibodies for Binding to Recombinantgp120 and gp41 by ELISA Assay

Recombinant gp120 with sequence derived from gp120 of primary HIV-1isolate JR-CSF and expressed in insect cells was obtained from IAVI NACrepository. Recombinant gp41 generated with sequences derived from HxB2clone of HIV-1 and expressed in Pichia pastoris was manufactured byVybion, Inc., obtained from IAVI NAC repository Sheep anti-gp120antibodies used as capturing agent to indirectly immobilize gp120 onELISA plates was purchased from Aalto Bio Reagents (Dublin, Ireland).All ELISA assays were conducted at 25 μL/well on MaxiSorp plates fromNunc.

In anti-gp120 ELISA, recombinant gp120 (0.5 μg/ml) was captured on 384well ELISA plates pre-coated (at 4° C. overnight) with goat anti-gp120(5 μg/ml) in BSA-containing assay buffer (PBS with 0.05% Tween-20) for 1hr at room temperature. After excess gp120 was removed and plates werewashed thrice with assay buffer, B cell culture supernatants diluted5-fold was added to incubate for 1 hr at room temperature. Followingthree washes in assay buffer, secondary HRP-conjugated goat anti-humanIg Fc in BSA-containing assay buffer was added and incubated for about 1hr at room temperature. 3,3′,5,5′-tetramethylbenzidine (TMB) substratewas used to develop the colorimetric readouts after washing the ELISAplates 3 times.

For anti-gp41 ELISA, recombinant gp41 was directly immobilized on 384well ELISA plates by adding 1 μg/ml and incubating at 4° C. overnight,followed by blocking with BSA-containing assay buffer. The rest of theassay protocol was similar to that for anti-gp120 ELISA.

Hits from the ELISA assay were identified in a singlet screen based onoptical density (OD) values above 3× assay background. A serialtitration standard curve of control antibody was included on each plate.

Example 4 Neutralization Assay for Screening Antibodies AgainstPseudotyped HIV Viruses

The neutralization assay approach has been described previously (BinleyJ M, et al., (2004). Comprehensive Cross-Clade Neutralization Analysisof a Panel of Anti-Human Immunodeficiency Virus Type 1 MonoclonalAntibodies. J. Virol. 78: 13232-13252) and was modified and standardizedfor implementation in 384-well format.

Neutralization by monoclonal antibodies and patient sera was performedusing a single round of replication pseudovirus assay. (Richman, D. D.,et al. Proc Natl Acad Sci USA 100, 4144-4149 (2003)). Pseudovirusneutralization assays were performed using HIV-1_(JR-CSF) alaninemutants as described in Pantophlet, R., et al. J Virol 77, 642-658(2003). Neutralization activity was measured as a reduction in viralinfectivity compared to an antibody-free control using a TZM-BL assay.(Li, M., et al. J Virol 79, 10108-10125 (2005)). Monoclonal antibodyneutralization assays using phytohaemgglutinin-activated peripheralblood mononuclear cells (PBMC) isolated from three healthy human donorsas target cells were performed as described in Scarlatti, G. et al,(1993) J. Infect. Dis. 168:207-210; Polonis, V. et al, (2001) AIDS Res.Hum. Retroviruses 17:69-79. Memory B cell supernatants were screened ina micro-neutralization assay against HIV-1_(SF162), HIV-1_(JR-CSF), andSIV_(mac239) (negative control). This assay was based on the 96-wellpseudotyped HIV-1 neutralization assay (Monogram Biosciences) and wasmodified for screening 15 μl B cell culture supematants in a 384-wellformat.

Pseudotyped virus from SF162 and JR-CSF isolates of HIV-1 and SIV mac239(control virus) were generated by co-transfecting Human Embryonic Kidney293 cells (293 cells) with 2 plasmids encoding the Envelope cDNAsequence and the rest of the HIV genome separately. In the HIV genomeencoding vector, the Env gene was replaced by the firefly luciferasegene. Transfectant supernatants containing pseudotyped virus wereco-incubated overnight (18 hours) with B cell supernatants derived fromactivation of an infected donor's primary peripheral blood mononuclearcells (PBMCs). U87 cells stably transfected with and expressing CD4 plusthe CCR5 and CXCR4 coreceptors were added to the mixture and incubatedfor 3 days at 37° C. Infected cells were quantified by luminometry.SIVmac239 was used as the negative control virus.

The neutralization index was expressed as the ratio of normalizedrelative luminescence units (RLU) of the test viral strain to that ofthe control virus SIVmac239 derived from the same test B cell culturesupernatant. The cut-off values used to distinguish neutralizing hitswere determined by the neutralization index of a large number of“negative control wells” containing B cell culture supernatants derivedfrom healthy donors. The false positive rate using the cut-off value of1.5 was very low (1-3%; FIG. 5A), and it was reduced to zero if thecut-off value of 2.0 was used (FIG. 5B).

FIG. 5 summarizes the screening results from which B cell cultures wereselected for antibody rescue and the monoclonal antibodies 1496_C09(PG9), 1443_C16 (PG16), 1456_P20 (PG20), 1460_G14 (PGG14), and 1495_C14(PGC14) were derived. The results reveal that the majority ofneutralizing B cell culture supernatants did not have binding reactivityto soluble recombinant gp120 or gp41 proteins.

Table 15 shows the screening results of the monoclonal antibodies1496_C09 (PG9), 1443_C16 (PG16), 1456_P20 (PG20), 1460_G14 (PGG14), and1495_C14 (PGC14) during the course of their identification in the methoddescribed in this invention. The neutralization activity of eachantibody and its corresponding binding reactivity to soluble recombinantgp120 or gp41, in the context of B cell culture supernatant andrecombinant transfectant supernatants are illustrated.

TABLE 15

Lightest grey: suggested H &L pair for monoclonal antibody per prioritywell. Medium grey with black lettering: Denotes clones derived from samerecombinant H or L chain pool of the priority well with identicalsequences. Bolded: 1496 C09 λ3 clone 024 is likely a cross-contaminantin the recombinant DNA pool as it is identical to 1443 C16 λ2 019 insequence. 1496 C09 λ2 017 sequence represents 21/22 clones in the pool.*Anti-gp120 and anti-gp41 concentrations were extrapolated from b12 and2F5 standard curves in quantitative ELISA, respectively. N/A = notapplicable because these hits were neither gp-120- nor gp-41 positive inB cell culture. ND = not done.

The purified monoclonal antibodies 1496_C09 (PG9), 1443_C16 (PG16),1456_P20 (PG20), 1460_G14 (PGG14), and 1495_C14 (PGC14) were tested forneutralization of 6 additional HIV strains from clades A (94UG103), B(92BR020, JR-CSF), C (93IN905, IAVI_C22), and CRF01_AE (92TH021) (Table16). The antibodies 1496_C09 (PG9), 1443_C16 (PG16) and 1495_C14 (PGC14)showed neutralization profile similar to that obtained with the donorsera neutralization profile. The pseudoviruses were preincubated witheach monoclonal antibody for 1 hour or 18 hours prior to the infectionof target cells. IC₅₀ values derived from 1 or 18 hours preincubationwere similar. Therefore, in further neutralization assays testingpurified monoclonal antibodies, 1 hour of preincubation was used.

Table 17A shows the neutralization profiles for the 5 monoclonalantibodies 1496_C09 (PG9), 1443_C16 (PG16), 1456_P20 (PG20), 1460_G14(PGG14), and 1495_C14 (PGC14) in IC₅₀ values on an extended panel of 16pseudoviruses, together with known cross-clade neutralizing antibodiesb12, 2G12, 2F5 and 4E10. Table 17B shows the IC₉₀ of two monoclonalantibodies, 1443_C16 (PG16) and 1496_C09 (PG9) on the same expandeddiverse panel of 16 HIV pseudoviruses from different clades, togetherwith known cross-clade neutralizing antibodies b12, 2G12, 2F5 and 4E10.FIG. 4 shows neutralization activity of monoclonal antibodies 1443_C16(PG16) and 1496_C09 (PG9) to 3 other pseudoviruses not included in Table16.

TABLE 16 Neutralizing Antibody Assay: IC50 Summary Virus/Ab IC50 (ug/mL)Except Where Noted incubation SF162 94UG103 92BR020 93IN905 IAVI_C2292TH021 JRCSF NL43 aMLV 1 hour 1443C16 >50 0.0211 >50 0.3302*** 0.1143*0.1362*** <0.0025 <0.0025** >50 18 hour 1443C16 >50 0.0085 >50 0.2553*0.1064*** 0.0435 <0.0025 4.9874** >50 1 hour 1456P200.1946 >50 >50 >50 >50 >50 >50 0.20 >50 18 hour 1456P20 0.0661 >50 >503.8384* >50 >50 >50 0.05 >50 1 hour 1460G140.1789 >50 >50 >50 >50 >50 >50 0.17 >50 18 hour 1460G14 0.0573 >50 >503.1738* >50 >50 >50 0.05 >50 1 hour 1495C14 0.0069 >501.1697 >50 >50 >50 >50 0.35 >50 18 hour 1495C14 <0.0025 >50 0.24420.1458* 13.3798 >50 >50 0.15 >50 1 hour 1496C09 >50 0.3336 >50 0.144424.8611 0.0612 <0.0025 0.2944* >50 18 hour 1496C09 >50 0.0942 >50 0.06192.1073 0.0571 <0.0025 38.03 >50 1 hour Z23 (1/dil'n) 13521 188 616 369340 175 438 4793 <100 18 hour Z23 (1/dil'n) 66074 262 1292 1396 614 3361054 9472 <100 *plateau **flat inhibition curve - probably <0.0025 withplateau ***very long, shallow slope ****plateau with very long, shallowslope to curve

TABLE 17A Neutralization Profile on a Diverse Panel of Viruses: IC₅₀Values PG9 PG16 PGC14 PGG14 PG20 b12 2G12 2F5 4E10 Clade A 94UG1030.1731 0.0080 >50 >50 >50 3.54 >50 3.79 9.7 92RW020 0.0637 0.0040****28.5960 >50 >50 >50 0.56 3.37 3.38 93UG077 >50 >50 >50 >50 >50 41.12 >504.45 11.15 Clade B 92BR020 >50 >50 0.6366 >50 >50 27.5 2.26 >50 41.44APV-13 >50 >50 >50 >50 >50 >25 23.9 2.8 3.8 APV-1726.4465 >50 >50 >50 >50 >25 >50 2 5.1 APV-6 0.0869 0.08**** 7.4062 >5025.7798 >25 5.3 0.1 0.4 JRCSF <0.0025 <0.0025 >50 >50 >50 0.16 0.66 3.366 Clade C 93IN905 0.1400 0.1016*** >50 >50 >50 34.15 >50 >50 1.55IAVI-C18 0.0535 0.0067 >50 >50 >50 >50 >50 >50 >50 IAVI-C22 24.86000.0687* 9.4999 >50 >50 3.6042 >50 >50 1.0229 IAVI-C3 12.910314.8372 >50 >50 >50 5.0000 >50 >50 5.0000 Clade D 92UG02410.9552 >50 >50 >50 >50 49.06 0.59 1.27 1.3292UG005 >50 >50 >50 >50 >50 >50 >50 11.75 8.66 CRF01_AE 92TH021 0.11050.1273*** >50 >50 >50 9.90 >50 1.51 1.9 CMU02 >50 >50 >50 >50 >504.25 >50 0.38 0.59 Pos C NL43 N/A <0.0025** 0.3727 0.1717 0.1880 0.060.75 2.41 4.95 Neg C aMLV >50 >50 >50 >50 >50 >50 >50 >50 >50 NA—NotApplicable IC₅₀: Inhibitory concentration to inhibit 50% of the virus

TABLE 17B Neutralization Profile on a Diverse Panel of Viruses: IC₉₀Values for mAbs PG9 and PG16. PG9 PG16 b12 2G12 2F5 4E10 Clade A 94UG1033.3736 1.5915 47.29 >50 46.63 >50 92RW020 6.5462 >50 >50 6.23 27.7436.11 93UG077 >50 >50 >50 >50 33.44 >50 Clade B 92BR020 >50 >50 >5024.09 >50 >50 APV-13 >50 >50 >50 N/A N/A N/A APV-17 >50 >50 >50 N/A N/AN/A APV-6 1.9591 44.2600 >50 N/A N/A N/A JRCSF <0.0025 0.0130 1.17 5.3825.31 44.07 Clade C 93IN905 1.8945 >50 >50 >50 >50 12.82 IAVI-C18 0.86590.2074 >50 >50 N/A >50 IAVI-C22 >50 >50 29.6187 >50 >50 16.405IAVI-C3 >50 >50 >50 N/A N/A Clade D 92UG024 >50 >50 >50 7.57 34.44 23.7192UG005 >50 >50 >50 >50 >50 >50 CRF01_AE 92TH021 1.9871 23.4110 >50 >5018.78 23.52 CMU02 >50 >50 34.2 >50 12.25 13.4 Pos C NL43 N/A >50 0.2815.75 19.32 29.56 Neg C aMLV >50 >50 >50 >50 >50 >50 NA—Not ApplicableIC₉₀: inhibitory concentration to inhibit 90% of the virus ***Plateaueffect

Example 5 Binding Specificity of Monoclonal Antibodies for HIV gp120 byELISA Assay

The purified anti-gp120 monoclonal antibodies, 1456_P20 (PG20), 1460_G14(PGG14), and 1495_C14 (PGC14), were confirmed for binding reactivity togp120 in ELISA assays. When titrated in serial dilutions, all threeantibodies exhibited similar binding profiles that suggest significantlyhigher relative avidity than control anti-gp120 (b12). MAb b12 isdirected against an epitope overlapping the CD4 binding site. (Burton DR et al. 1994. Efficient neutralization of primary isolates of HIV-1 bya recombinant human monoclonal antibody. Science 266:1024-1027).

FIG. 5 shows dose response curves of 1456_P20 (PG20), 1460_G14 (PGG14),and 1495_C14 (PGC14) binding to recombinant gp120 in ELISA as comparedto control anti-gp120 (b12). Data shown represented average OD values oftriplicate ELISA wells obtained on the same plate.

The monoclonal antibodies 1443_C16 (PG16) and 1496_C09 (PG9) were testedfor binding to soluble recombinant envelope proteins derived fromseveral HIV strains in ELISA assay. ELISA assays were performed asdescribed in Pantophlet, R., et al. J Virol 77, 642-658 (2003). Forantigen binding ELISAs, serial dilutions of PG9 were added to antigencoated wells and binding was probed with alkaline phosphatase-conjugatedgoat anti-human immunoglobulin G (IgG) F(ab′)2 Ab (Pierce). Forcompetition ELISAs, competitor mAbs were added to ELISA wells andincubated for 15 min prior to adding 15 μg/mL biotinylated PG9 to eachwell. Biotinylated PG9 was detected using alkaline phosphataseconjugated streptavidin (Pierce) and visualized using p-nitrophenolphosphate substrate (Sigma). HIV-HXB2 gp120 was used for competitionELISA assays.

FIG. 6 shows results from ELISA binding assays of monoclonal antibodies1443_C16 (PG16) and 1496_C09 (PG9) to HIV-1 YU2 gp140, JR-CSFgp120,membrane-proximal external regions (MPER) peptide of gp41 and V3polypeptide. Specificity of the monoclonal antibodies 1443_C16 (PG16)and 1496_C09 (PG9) for gp120 was then confirmed, but it was noted thatthe binding to soluble envelope glycoprotein was weak.

Example 6 Binding Reactivity of Monoclonal Antibodies 1443_C16 (PG16)and 1496_C09 (PG9) to Envelope Proteins Expressed on Transfected CellSurface and Competition by Soluble CD4 (sCD4)

MAb cell binding assays were performed as described in Pancera, M. &Wyatt, R. Virology 332, 145-156 (2005). Titrating amounts of PG9 andPG16 were added to HIV-1 Env transfected 293T cells, incubated for 1 hrat 4° C. washed with FACS buffer, and stained with goat anti-human IgGF(ab′)₂ conjugated to phycoerythin. For competition assays, competitorantibodies were added to the cells 15 min prior to adding 0.1 μg/mLbiotinylated PG9 or PG16. For sCD4 inhibition assays, 40 μg/mL sCD4 wasadded to the cells and incubated for 1 h at 4° C. prior to addingtitrating amounts of antibodies. Binding was analyzed using flowcytometry, and binding curves were generated by plotting the meanfluorescence intensity of antigen binding as a function of antibodyconcentration.

Ninety-six-well ELISA plates were coated overnight at 4° C. with 50 μLPBS containing 100 ng gp120 or gp140 per well. The wells were washedfour times with PBS containing 0.025% Tween 20 and blocked with 3% BSAat room temperature for 1 h. Serial dilutions of PG9 were added toantigen coated wells, incubated for 1 h at room temperature, and washed4× with PBS supplemented with 0.025% Tween 20. Binding was probed withalkaline phosphatase-conjugated goat anti-human immunoglobulin G (IgG)F(ab′)2 Ab (Pierce) diluted 1:1000 in PBS containing 1% BSA and 0.025%Tween 20. The plate was incubated at room temperature for 1 h, washedfour times, and the plate was developed by adding 50 μL of alkalinephosphatase substrate (Sigma) to 5 mL alkaline phosphatase stainingbuffer (pH 9.8), according to the manufacturer's instructions. Theoptical density at 405 nm was read on a microplate reader (MolecularDevices). For competition ELISAs, competitor mAbs were added togp120_(HxB2) or gp140_(YU2) coated ELISA wells and incubated for 15 minprior to adding 15 μg/mL biotinylated PG9 to each well. Biotinylated PG9was detected using alkaline phosphatase conjugated streptavidin (Pierce)and visualized using p-nitrophenol phosphate substrate (Sigma). For sCD4inhibition ELISAs, 5 μg/mL sCD4 was added to antigen-coated wells andincubated for 15 min at room temperature prior to adding titratingamounts of PG9. A FACSArray™ plate reader (BD Biosciences, San Jose,Calif.) was used for flow cytometric analysis and FlowJo™ software wasused for data interpretation.

HIV gp160 derived from YU2 was transfected in 293 cells. Binding ofmonoclonal antibodies 1443_C16 (PG16) and 1496_C09 (PG9) were detectedin transfected cells (FIG. 7). The preincubation of transfected cellswith soluble CD4 (sCD4) partially inhibited binding of monoclonalantibody for 1496_C09 (PG9), and for 1443_C16 (PG16) suggesting theantibody binding is effected by the presence of sCD4. Binding isinhibited by at least 15%, at least 20%, at least 25%, or at least 30%.Binding of monoclonal antibodies 1443_C16 (PG16) and 1496_C09 (PG9) to293 cells transfected with gp160 derived from JR-CSF and ADA strains wasalso detected (FIG. 8). The binding of both monoclonal antibodies1443_C16 (PG16) and 1496_C09 (PG9) to JR-CSF transfected cells wasblocked by sCD4. Results further confirm that binding activities ofmonoclonal antibodies 1443_C16 (PG16) and 1496_C09 (PG9) are affected bythe presence of sCD4.

Example 7 Binding Reactivity of Monoclonal Antibodies 1443_C16 (PG16)and 1496_C09 (PG9) to Pseudoviruses

In vitro virus capture assay was used to test if monoclonal antibodies1443_C16 (PG16) and 1496_C09 (PG9) bind to intact entry competentpseudoviruses. The monoclonal antibodies 1443_C16 (PG16) and 1496_C09(PG9) were coated at the bottom of 96-well plate via anti-human Fc.JR-CSF pseudovirus was added and captured by the monoclonal antibody1443_C16 (PG16) or 1496_C09 (PG9) in a dose dependent manner. Targetcells were added to initiate infection. Infection measured in RLU thenrepresented the binding and capture activity of monoclonal antibodies1443_C16 (PG16) and 1496_C09 (PG9). FIG. 9 shows the binding and captureof JR-CSF pseudovirus by both monoclonal antibodies 1443_C16 (PG16) and1496_C09 (PG9) in a dose dependent manner, which is similar or betterthan another known broad and potent neutralizing antibody 2G12.

Example 8 Monoclonal Antibodies 1443_C16 (PG16) and 1496_C09 (PG9)Cross-Compete with Each Other and with sCD4 in Binding to JR-CSFPseudovirus

In a competition version of virus capture assay where JR-CSF pseudoviruswas captured by monoclonal antibodies 1443_C16 (PG16), competition ofthe capture by either monoclonal antibodies 1443_C16 (PG16), 1496_C09(PG9) and sCD4 was measured. FIG. 10B shows that binding of monoclonalantibody 1443_C16 (PG16) to JR-CSF pseudovirus was blocked by itself,monoclonal antibody 1496_C09 (PG9) and sCD4 in a dose dependent manner.In a corresponding manner, FIG. 10B shows that binding of monoclonalantibody 1496_C09 (PG9) to JR-CSF pseudovirus was blocked by itself,monoclonal antibody 1443_C16 (PG16) and sCD4 in a dose dependent manner.Results indicated that the monoclonal antibodies 1443_C16 (PG16) and1496_C09 (PG9) bind to closely related epitopes on gp120 and theirbinding is affected by the presence of sCD4 presumably due toconformational changes induced on HIV-1 envelope by sCD4.

Example 9 Antigen Binding Properties of PG9 and PG16

Antigen binding properties of PG9 and PG16 were determined by ELISAassays as shown in FIG. 11A-B. Binding of PG9 and PG16 to monomericgp120 and artificially trimerized gp140 constructs were determined (FIG.11A). Binding of PG9 and PG16 to Env expressed on the surface of 293Tcells as determined by flow cytometry. (FIG. 11B). b12 was used as acontrol for ELISA assays. The bNAb b12 and the non-neutralizing antibodyb6 were included in the cell surface binding assays to show the expectedpercentages of cleaved and uncleaved Env expressed on the cell surface.

Example 10 Binding of PG9 and PG16 to Cleavage-Defective HIV-1_(YU2)Trimers

Binding of PG9 and PG16 to cleavage-defective HIV-1_(YU2) trimers wasdetermined by flow cytometry. PG9 and PG16 bind with high affinity tocleavage-defective HIV-1_(YU2) trimers as shown in FIG. 12. Bindingcurves were generated by plotting the mean fluorescence intensity (MFI)of antigen binding as a function of antibody concentration.

Example 11 Mapping the PG9 and PG16 Epitopes

Mapping the epitopes of PG9 and PG16 epitopes was performed by acompetitive binding assay as shown in FIG. 13. PG9 and PG16 competedwith each other for cell surface Env binding and neither antibodycompeted with the CD4bs antibody b12 for Env binding. Competitorantibody is indicated at the top of each graph. (FIG. 13A). Ligation ofcell surface Env with sCD4 diminished binding of PG9 and PG16. 2G12 wasincluded to control for CD4-induced shedding of gp120. (FIG. 13B). sCD4inhibited binding of PG9 to artificially trimerized gp140_(JR-CSF) asdetermined by ELISA. (FIG. 13C). PG9 competed with 10/76b (anti-V2),F425/b4e8 (anti-V3) and X5 (CD4i) for gp120 binding in competition ELISAassays. (FIG. 13D). PG9 and PG16 failed to bind variable loop deletedHIV-1_(JR-CSF) variants expressed on the surface of 293T cells. 2G12 wasincluded to control for cell surface Env expression. (FIG. 13E).

Example 12 Competition ELISA Assays Using PG9

When competition ELISA assays using PG9 were performed, PG9 competedwith c108g (anti-V2) and partially competed with 17b (CD4i). Nocompetition was observed with A32 (anti-C1/C2/C4/CD4i), C11 (C1), 2G12(glycan shield), b6 (CD4bs), b3 (CD4bs) or 23b (C1/C5) for gp120_(HxB2)binding as shown in FIG. 14.

Example 13 Binding of PG9 and PG16 to HIV-1_(JR-FL) E168K

Antibody binding to HIV-1JR-FLΔCT E168K Env expressed on the surface of293T cells as determined by flow cytometry is shown in FIG. 15. Acytoplasmic tail deleted construct was used to increase cell surfaceexpression. The bNAb b12 and the non-neutralizing antibody b6 wereincluded in the cell surface binding assays to show the expectedpercentages of cleaved and uncleaved Env expressed on the cell surface.(Pancera M., et al. Virology 332:145 (2005). HIV-1JR-FL E168K wasgenerated by site-directed mutagenesis. Binding curves were generated byplotting the MFI of antigen binding as a function of antibodyconcentration.

Example 14 PG9 Binding to Deglycosylated gp120

gp120_(DU422) was treated with 40 mU/μg Endoglycosidase H (Endo H, NewEngland Biolabs) in sodium acetate buffer for 24 hr at 37° C. Mocktreated gp120 was treated under same conditions, but the enzyme wasomitted from the reaction. Binding of PG9 and b6 to EndoH treated andmock treated gp120 was determined by ELISA as shown in FIG. 16.

Example 15 Neutralization Activity Against HIV-1_(SF162) K160N

Neutralization activity of PG9 and PG16 against HIV-1_(SF162) andHIV-1_(SF162) K160N was determined using a single-round replicationluciferase reporter assay of pseudotyped virus. HIV-1_(SF162) K160N wasgenerated by site-directed mutagenesis as shown in FIG. 17.

Example 16 Binding of PG9 and PG16 to Mixed Trimers

Alanine substitutions at positions 160 and 299 were introduced intoHIV-1_(YU2) Env to abolish binding of PG9 and PG16. An alaninesubstitution at position 295 was also introduced into the same constructto abrogate binding of 2G12. Co-transfection of 293T cells with WT andmutant plasmids in a 1:2 ratio resulted in the expression of 29% mutanthomotrimers, 44% heterotrimers with two mutant subunits, 23%heterotrimers with one mutant subunit, and 4% wild-type homotrimers.These proportions were calculated using the formula described in Yang,X., Kurteva, S., Lee, S., and J. Sodroski, J Virol 79(6):3500-3508(March 2005), and assumes that mutant and wild-type gp120s mix randomlyto form trimers. Binding of mAbs to Env trimers was determined by flowcytometry as shown in FIG. 18. b12 was included as control for Env cellsurface expression.

Example 17 PG9 or PG16 Neutralization Activity on HIV with AlanineMutations within gp120

Alanine mutations within gp120 of HIV decrease PG9 or PG16neutralization activity as shown in Table 21. In the table, amino acidnumbering is based on the sequence of HIV-1_(HxB2). Boxes are colorcoded as follows: white, the amino acid is identical among 0 to 49% ofall HIV-1 isolates; light grey, the amino acid is identical among 50 to90% of isolates; dark grey, the amino acid is identical among 90 to 100%of isolates. Amino acid identity was determined based on a sequencealignment of HIV-1 isolates listed in the HIV sequence database athiv-web.lanl.gov/content/hiv-db/mainpage.html. C refers to constantdomains and V refers to variable loops. Neutralization activity isreported as fold increase in IC₅₀ value relative to WT JR-CSF and wascalculated using the equation (IC₅₀ mutant/IC₅₀ WT). Boxes are colorcoded as follows: white, substitutions which had a negative effect onneutralization activity; light grey, 4-9 fold IC₅₀ increase; mediumgrey, 10-100 fold IC₅₀ increase; dark grey, >100 fold IC₅₀ increase.Experiments were performed in triplicate and values represent an averageof at least three independent experiments.

TABLE 18A

TABLE 18B

TABLE 18C

TABLE 18D

TABLE 18E

TABLE 18F

^(a)White squares indicate an IC50 of >50 μg/mL, black squares indicate50 μg/mL > IC50 > 10 μg/mL, lightest grey squares indicate 10 μg/mL >IC50 > 1 μg/mL, medium grey squares indicate 1 μg/mL > IC50 > 0.1 μg/mL,darker grey squares indicate IC50 < 0.01 μg/mL. N.D., not done.^(b)White squares indicate an IC50 of < 1:100 dilution, darkest greysquares indicate 1:50 > IC50 > 1:150, lightest grey squares indicate1:150 > IC50 > 1:500, medium grey squares indicate 1:500 > IC50 >1:1000, darker grey squares indicate IC50 > 1:1000 dilution.

TABLE 19A Neutralization Potency.

White boxes indicate a medium potency of >50 μg/mL, darkest grey between20 and 50 μg/mL, lightest grey between 2 and 20 μg/mL, medium greybetween 0.2 and 2 μg/mL, and darker grey < 0.2 μg/mL. *CRF_07BC andCRF_08BC viruses not included in the clade analysis because there wasonly one virus tested from each of these ciades.

TABLE 19B Neutralization Breadth.

White boxes indicate that no viruses were neutralized, darkest greyindicate 1 to 30% of viruses were neutralized, lightest grey indicate30% to 60% of viruses were neutralized, rnedium grey indicate 60 to 90%of viruses were neutralized, and darker grey indicate 90 to 100% ofviruses were neutralized. *CRF_07BC and CRF_08BC viruses not included inthe clade analysis because there was only one virus tested from each ofthese clades.

TABLE 20 Neutralization activity of PG9 and PG16 against JR-CSFpseudovirus containing alanine point mutations.

^(a)Amino acid number is based on the sequence of HIV-1_(HxB2).^(b)White boxes indicate that the amino acid is identical among 0 to 49%of all HIV isolates, light grey boxes indicate that the amino acid isidentical among 50-90% of all HIV isolates, and dark grey boxes indicatethat the amino acid is identical among 90-100% of all HIV isolates.Amino acid identity was determined based upon a sequence alignment ofHIV-1 isolates listed in the HIV sequence database athttp://hiv-gov/content/hiv-db/mainpage.html. ^(c)C refers to constantdomains and V refers to variable loops. ^(d)Neutralization activity isreported as fold increase in IC50 value relative to WT JR-CSF and wascalculated using the equation (IC50 mutant/IC50 WT). White:substitutions which had a negligible effect on neutralization activity,lightest grey: 4-9 fold IC50 increase, dark grey: 10-100 fold IC50increase, darkest grey: >100 fold IC50 increase. Experiments wereperformed in triplicate and values represent an average of at leastthree independent experiments.

TABLE 21 Alanine mutations that decrease PG9 and P16 neutralizationactivity.

^(a)Amino acid numbering is based on the sequence of HIV-1_(HxB2).^(b)Boxes are color coded as follows: white, the amino acid is identicalamong 0 to 49% of all HIV-1 isolates; light grey, the amino acid isidentical among 50 to 90% of isolates; dark grey, the amino acid isidentical among 90 to 100% of isolates. Amino acid identity wasdetermined based on a sequence alignment of HIV-1 isolates listed in theHIV sequence database athttp://hiv-web.lanl.gov/content/hiv-db/mainpage.html. ^(c)C refers toconstant domains and V refers to variable loops. ^(d)Neutralizationactivity is reported as fold increase in IC50 value relative to WTJR-CSF and was calculated using the equation (IC₅₀ mutant/IC₅₀ WT).Boxes are color coded as follows: white, substitutions which had anegative effect on neutralization activity; light grey, 4-9 fold IC₅₀increase; medium grey, 10-100 fold IC₅₀ increase; dark grey, >100 foldIC₅₀ increase. Experiments were performed in triplicate and valuesrepresent an average of at least three independent experiments.

Example 18 Identification of 14443 C16 (PG16) Sister Clones

1443 C16 sister clones were identified by screening clonal transfectionof rescued variable region genes for JR-CSR neutralization. Thus,antibodies that were identified as sister clones of 1443 C16 (PG16) havethe similar HIV neutralization profiles as the human monoclonal 1443 C16(PG16). Moreover, the nucleic acid or amino acid sequences of the sisterclone antibodies are at least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%,95%, 99%, 100% or any percentage point in between, identical to those of1443 C16 (PG16).

TABLE 22 1443 C16 Antibody JRCSF Sister mAbs Gamma Chain Cione LightChain Cione concentration (μg/ml) Neutralization index 1456 A121456_A12_G3_01_002 1456_A12_L2_01_023 0.006 0.90 1456_A12_L2_01_0360.012 0.82 1456_A12_L2_01_040 0.016 2.79 1456_A12_G3_01_0041456_A12_L2_01_023 <0.005 1.00 1456_A12_L2_01_036 <0.005 1.021456_A12_L2_01_040 0.005 6.95 1469 M23 1469_M23_G3_01_0051469_M23_L2_01_001 2.624 215.74 1469_M23_G3_01_006 0.000 10.05 1480 I081480_I08_G3_01_012 1480_I08_L2_01_005 <0.005 10.34 1480_I08_G3_01_016 10223.14 1480_I08_G3_01_021 <0.005 2.98 1480_I08_G3_01_032 <0.005 3.831480_I08_G3_01_037 34 1.36 1489_I08_G3_01_055 <0.005 1.16 1489 I131480_I13_G3_01_003 1489_I13_L2_01_007 0.0000 2.02 1480_I13_G3_01_0040.0009 22.86 1480_I13_G3_01_007 1.455 139.35 1503 H05 1503_H05_G1_01_0011503_H05_L2_01_021 0.013 0.96 1503_H05_G1_01_006 0.000 3.751503_H05_G1_01_005 1.108 91.41 1503_H05_G1_01_007 0.587 155.54 Note thatthe constant region of the 1456_A12 heavy chain clones used intransfection contains an error generated during the cloning process thatlead to no full-length IgG production.

Example 19 1443 C16 (PG16) Antibody Sister Clones and the 1443 C16(PG16) Antibody Exhibit Similar Neutralization Specificity

Antibodies 1456 A12, 1503 H05, 1489 I13 and 1469 M23 were tested forneutralization activity against several pseudoviruses containingdistinct mutations that map the reactivity epitope of 1443 C16 (PG16) ongp120 in a standard TZM-b1 assay (Table 23). Like 1443 C16 (PG16), whichdoes not bind or neutralize wild-type JR-FL, but instead, neutralizesJR-FL with the E168K mutation, all 1443 C16 (PG16) sister clonesneutralize JR-FL(E168K) with low IC50 values. Similarly, all 1443 C16(PG16) sister clones do not neutralize the Y318A mutants and 1309Amutants of JR-CSF, where the part of the putative binding epitope ismapped on the V3 tip.

TABLE 23 Neutralization specificity of 1443 C16 (PG16) sister clones asshown with specific mutations on gp120. IC50 (ug/ml) mAb JR-CSFJR-CSF(Y318A) JR-CSF(I309A) JR-FL(E168K) ADA 92RW020 1503 H050.001 >1.0 >1.0 0.002 0.003 0.020 1456 A12 0.001 >1.0 >1.0 0.003 0.0050.050 1469 M23 0.002 >1.0 >1.0 0.005 0.005 0.050 1489 I130.002 >1.0 >1.0 0.005 0.008 0.030 1443 C16 0.001 >1.0 >1.0 0.006 0.0040.090 1496 C09 0.006 0.001 0.001 0.020 0.200 0.100

Example 20 1443 C16 (PG16) Sister Clones Exhibit Similar NeutralizationBreadth and Potency as 1443 C16 (PG16) for Clade B and Clade C Viruses

The antibodies 1456 A12, 1503 H05, 1489 I13 and 1469 M23 exhibitneutralization activity against a panel of clade B and clade Cpseudoviruses with similar breadth as does 1443 C16 (PG16) in a standardTZM-b1 assay (Table 24). The neutralization potency of each sister clonefor each pseudovirus is comparable to that for 1443 C16 (PG16). When theIC50 value is determined, the value for the sister clone is within a 0.5log range from that for 1443 C16 (PG16).

TABLE 24 Neutralization breadth and potency of 1443 C16 (PG16) sisterclones. IC50 (ug/ml) 1443 1456 1469 1503 1489 Virus C16 A12 M23 H05 I13Clade B CAAN 6.37 10.61 17.72 13.46 24.87 REJ04541 <0.01 <0.01 0.39 0.220.34 THRO.18 2.19 2.08 7.01 4.12 7.41 PVO.4 12.3 10.42 21.25 11.01 20.57TR0.11 3.61 3.05 7.52 4.30 10.94 AC10 <0.01 <0.01 <0.01 <0.01 <0.01Clade C DU156 <0.01 <0.01 <0.01 <0.01 <0.01 DU422 0.59 0.36 0.97 0.711.87 Du172 <0.01 <0.01 <0.01 <0.01 <0.01 ZM214 >25 >25 >25 >25 >25 ZM233<0.01 <0.01 <0.01 <0.01 <0.01 CAP45 <0.01 <0.01 <0.01 <0.01 <0.01 ZM249<0.01 <0.01 <0.01 <0.01 <0.01 Control MuLV >25 >25 >25 >25 >25

Other Embodiments

Although specific embodiments of the invention have been describedherein for purposes of illustration, various modifications may be madewithout deviating from the spirit and scope of the invention.Accordingly, the invention is not limited except as by the appendedclaims. While the invention has been described in conjunction with thedetailed description thereof, the foregoing description is intended toillustrate and not limit the scope of the invention, which is defined bythe scope of the appended claims. Other aspects, advantages, andmodifications are within the scope of the following claims.

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. All UnitedStates patents and published or unpublished United States patentapplications cited herein are incorporated by reference. All publishedforeign patents and patent applications cited herein are herebyincorporated by reference. Genbank and NCBI submissions indicated byaccession number cited herein are hereby incorporated by reference. Allother published references, documents, manuscripts and scientificliterature cited herein are hereby incorporated by reference.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is:
 1. An isolated fully human anti-HIV-1 PG9 monoclonalantibody comprising (a) a light chain variable region comprisingcomplementarity determining regions having the amino acid sequences ofSEQ ID NOS: 45, 126 and 127 and (b) a heavy chain variable regioncomprising complementarity determining regions having the amino acidsequences of SEQ ID NOS: 7, 123 and
 124. 2. An isolated fully humananti-HIV-1 PG16 monoclonal antibody comprising (a) a light chainvariable region comprising complementarity determining regions havingthe amino acid sequences of SEQ ID NOS: 41, 92, and 95 and (b) a heavychain variable region comprising complementarity determining regionshaving the amino acid sequences of SEQ ID NOS: 6, 88 and
 89. 3. Anisolated fully human anti-HIV-1 PG9 monoclonal antibody comprising (a) alight chain variable region comprising amino acid sequence SEQ ID NO: 40and (b) a heavy chain variable region comprising amino acid sequence ofSEQ ID NO:
 28. 4. An isolated fully human anti-HIV-1 PG16 monoclonalantibody comprising (a) a light chain variable region comprising aminoacid sequence SEQ ID NO: 14 and (b) a heavy chain variable regioncomprising amino acid sequence of SEQ ID NO: 59 or 65 or (a) a lightchain variable region comprising amino acid sequence SEQ ID NO: 47 and(b) a heavy chain variable region comprising amino acid sequence of SEQID NO: 50 or (a) a light chain variable region comprising amino acidsequence SEQ ID NO: 56 and (b) a heavy chain variable region comprisingamino acid sequence of SEQ ID NO: 53 or (a) a light chain variableregion comprising amino acid sequence SEQ ID NO: 142 and (b) a heavychain variable region comprising amino acid sequence of SEQ ID NO: 139.5. An isolated fully human anti-HIV-1 PG9 monoclonal antibody comprisinga heavy chain sequence of SEQ ID NO.: 39 and a light chain sequence ofSEQ ID NO.:
 30. 6. An isolated fully human anti-HIV-1 PG16 monoclonalantibody comprising a heavy chain sequence of SEQ ID NO.: 31 and a lightchain sequence of SEQ ID NO.:
 32. 7. A composition comprising theantibody of any one of claims 1 to
 6. 8. A pharmaceutical compositioncomprising the antibody of any one of claims 1 to 6 and apharmaceutically acceptable carrier.